WO2019192182A1 - Application of schizochytrium limacinum and preparation thereof in improvement of quality and yield of animal product - Google Patents

Application of schizochytrium limacinum and preparation thereof in improvement of quality and yield of animal product Download PDF

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Publication number
WO2019192182A1
WO2019192182A1 PCT/CN2018/115327 CN2018115327W WO2019192182A1 WO 2019192182 A1 WO2019192182 A1 WO 2019192182A1 CN 2018115327 W CN2018115327 W CN 2018115327W WO 2019192182 A1 WO2019192182 A1 WO 2019192182A1
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Prior art keywords
animal
product
preparation
schizochytrium limacinum
dha
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PCT/CN2018/115327
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French (fr)
Chinese (zh)
Inventor
陈礼毅
钟惠昌
陈水荣
Original Assignee
厦门汇盛生物有限公司
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Priority claimed from CN201810297773.9A external-priority patent/CN109082381B/en
Priority claimed from CN201810300876.6A external-priority patent/CN109077194B/en
Priority claimed from CN201810300872.8A external-priority patent/CN109077193B/en
Application filed by 厦门汇盛生物有限公司 filed Critical 厦门汇盛生物有限公司
Priority to EP18913727.6A priority Critical patent/EP3756472A4/en
Priority to AU2018417303A priority patent/AU2018417303A1/en
Priority to JP2020529495A priority patent/JP7191102B2/en
Priority to US17/043,468 priority patent/US11583566B2/en
Publication of WO2019192182A1 publication Critical patent/WO2019192182A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/111—Aromatic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/116—Heterocyclic compounds
    • A23K20/121—Heterocyclic compounds containing oxygen or sulfur as hetero atom
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C12N1/125—Unicellular algae isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae

Definitions

  • the invention relates to the application of the genus Schizophyllum and its preparation in improving the quality and yield of animal products in agricultural organisms.
  • DHA docosahexaenoic acid
  • ⁇ -3 PUFAs polyunsaturated fatty acids
  • the DHA content in ordinary milk is extremely low, which is difficult to meet people's daily needs.
  • the addition of DHA to dairy products has more materials and consumes more production costs.
  • the addition process is likely to cause DHA price reduction, decomposition or odor.
  • polyunsaturated fatty acids such as DHA in ruminant diets
  • the DHA content in milk and muscle tissue can be increased, but the special digestive structure of ruminants makes most of the unsaturated fatty acids such as DHA enter the rumen and be mostly Conversion to saturated fatty acids greatly reduces the utilization of polyunsaturated fatty acids such as DHA.
  • the fatty acid rumen protection technology on the market mainly includes coating, hydrogenation and calcification.
  • PS Phosphatidylserine
  • the structure of PS determines its unique amphiphilic nature.
  • the negatively charged end is hydrophilic (or water soluble) and the other end is composed of fatty acids. It is lipophilic (or fat soluble).
  • DHA binds to the 2 position of phosphatidylserine glycerol backbone, DHA is more stable and more easily passes through the blood-brain barrier.
  • DHA and PS are absorbed in the form of 2-DHA-PS (ie, Sn-2 position DHA) in vitro, and finally converted into DHA-PS in the brain for neuroprotection.
  • 2-DHA-PS can have both DHA and The biological function of PS. Therefore, how to increase the content of DHA in eggs, enrich the source of different types of DHA, and expand the application field and consumption range of DHA is a problem to be solved.
  • the technical problem to be solved by the present invention is how to improve the quality and yield of animal products.
  • the present invention first provides any of the following applications of Schizochytrium limacinum or a preparation thereof:
  • the Schizochytrium limacinum may be Schizochytrium limacinum HS01, and the preservation number of the Schizochytrium limacinum HS01 in the General Microbiology Center of the Chinese Collection of Microorganisms and Cultures is CGMCC. No. 13746.
  • the active ingredient of the formulation may be the Schizochytrium limacinum.
  • the improving the quality of the animal product may be to increase the DHA content of the animal product and/or increase the DHA content of the animal product at the Sn-2 position and/or reduce the cholesterol content of the animal product.
  • the preparation may be a cracked algae powder.
  • the preparation may be prepared according to a method comprising the following steps (the method is referred to as a preparation method of a Schizochytrium preparation): cultivating the Schizochytrium limacinum to obtain a fermentation broth; using the fermentation broth The formulation was prepared.
  • the culture of the Schizochytrium limacinum can be carried out using a fermentation medium consisting of a solvent and a solute, the solvent being water, the solute and its concentration being glucose 60-150 g, respectively.
  • the starch may be corn starch or sodium starch octenyl succinate
  • the protein powder may be pea protein powder or whey protein powder.
  • the pH of the fermentation medium may specifically be 6.
  • Pea protein powder is the total protein of pea extracted from peas.
  • Whey protein powder is the total protein of milk extracted from milk.
  • the solute of the fermentation medium and its concentration may specifically be as follows: n1) or n2) or n3) or n4):
  • glucose 150g/L glucose 150g/L, yeast extract 25g/L, yeast powder 8g/L, Na 2 SO 4 20g/L, KCl 1.5g/L, MgSO 4 3.0g/L, K 2 SO 4 2.5g/L, KH 2 PO 4 2.0g/L, (NH 4 ) 2 SO 4 5.0g/L, CaCl 2 2.5g/L, CuSO 4 0.02g/L, ZnSO 4 0.02g/L, biotin 0.06g/L, corn starch 10g / L and pea protein powder 20g / L;
  • glucose 60g/L yeast extract 8g/L, yeast powder 3g/L, Na 2 SO 4 5g/L, KCl 0.5 g/L, MgSO 4 1.0g/L, K 2 SO 4 0.5g/L, KH 2 PO 4 1.0g/L, (NH 4 ) 2 SO 4 2.0g/L, CaCl 2 0.5g/L, CuSO 4 0.001g/L, ZnSO 4 0.001g/L, biotin 0.001g/L and octene Sodium succinate sodium starch 0.1g / L;
  • the preparing the preparation by using the fermentation liquid may include: drying the fermentation liquid to obtain the preparation.
  • the above method may further comprise adding an antioxidant to the fermentation broth after obtaining the fermentation broth, and then drying to obtain the cleavage algae powder (ie, the preparation).
  • the antioxidant can be an oil soluble antioxidant and/or a water soluble antioxidant.
  • the oil soluble antioxidant may be rosemary, natural mixed tocopherol, tea polyphenols and/or ascorbyl palmitate.
  • the water soluble antioxidant can be phytic acid, ascorbic acid and/or isoascorbic acid.
  • the ratio between the components is not required, and can be adjusted according to specific needs.
  • the antioxidant may specifically be a mixed antioxidant composed of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid.
  • the ratio between the substances in the mixed antioxidant may be the following p1), p2), p3) or p4):
  • P1 mass ratio of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid is 20:2:10:10:2;
  • the mass ratio of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid is 80:2:40:40:8.
  • the drying can be spray drying or drum drying or freeze drying.
  • the above method may further comprise washing the Schizochytrium limacinum in the fermentation broth.
  • the above method may further comprise adding the antioxidant to the washed Schizochytrium limacinum and then drying.
  • the amount of dissolved oxygen in the culture may be 0 to 80% (e.g., 10 to 80%).
  • the temperature of the culture may be 20 to 30 °C.
  • the culture time may be 72 to 120 hours.
  • the animal may be a1) or a2) or a3):
  • the animal product can be an egg produced by the animal.
  • the animal may be b1) or b2):
  • the cow can be a cow.
  • the cow may be a Holstein cow.
  • the animal product can be the milk of the animal, such as milk.
  • the preparation method of the Schizochytrium preparation is also within the scope of protection of the present invention.
  • Y1 a medium for cultivating the Schizochytrium limacinum, which is the fermentation medium;
  • the present invention also provides a method for improving the quality of an animal product, which comprises: feeding an animal Schizochytrium limacinum or a preparation thereof to improve the quality of the animal product.
  • the Schizochytrium limacinum may be the Schizochytrium limacinum HS01.
  • the active ingredient of the formulation may be the Schizochytrium limacinum.
  • the improving animal product quality may be c1) and/or c2) and/or c3):
  • the animal may be a ruminant, and the Schizochytrium limacinum or the preparation thereof may be fed in any one of d1)-d7):
  • the animal may be a poultry, and the mass of the Schizochytrium limacinum or the preparation thereof may be any one of e1)-e3) in the food to which the animal is fed:
  • the animal is a poultry whose food consists of a basal diet and the Schizochytrium limacinum or a preparation thereof.
  • the basal diet may be a corn-soybean meal type diet.
  • the ruminant may be a cow.
  • the cow can be a cow.
  • the cow may be a Holstein cow.
  • the animal product can be the milk of the animal, such as milk.
  • the poultry may be a2) or a3):
  • the animal product can be an egg produced by the animal.
  • the preparation can be prepared by the preparation method of the cleavage algae preparation.
  • an animal product prepared by the method for improving the quality of an animal product or a product obtained by processing the animal product is also within the scope of the present invention to prepare an animal product prepared by the method for improving the quality of an animal product or a product obtained by processing the animal product.
  • the animal is b1) or b2):
  • the animal product is milk.
  • the product obtained by processing the animal product is any one of f1)-f6):
  • the animal is a1) or a2) or a3):
  • the animal product is an egg.
  • the product obtained by processing the animal product is a native phospholipid DHA egg product.
  • the present invention also provides a method for increasing the yield of an animal product, the method comprising: feeding an animal Schizochytrium limacinum or a preparation thereof to increase the yield of the animal product.
  • the Schizochytrium limacinum may be the Schizochytrium limacinum HS01.
  • the active ingredient of the formulation may be the Schizochytrium limacinum.
  • the animal product can be an egg produced by the animal.
  • the preparation can be prepared by the preparation method of the cleavage algae preparation.
  • the animal is poultry, and the food of the animal is fed with any one of the mass content of the Schizochytrium limacinum or the preparation thereof, e1)-e3):
  • the poultry may be a2) or a3):
  • the preparation may further include a carrier.
  • the carrier can be a solid carrier or a liquid carrier.
  • the solid carrier may be a mineral material, a plant material or a polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth;
  • the plant material may be at least one of corn flour, soy flour, and starch;
  • the polymer compound may be polyvinyl alcohol and/or polyglycol.
  • the liquid carrier can be an organic solvent, vegetable oil, mineral oil or water; the organic solvent can be decane and/or dodecane.
  • the active ingredient may be present as a cultured living cell, a fermentation broth of a living cell, a filtrate of a cell culture, or a mixture of a cell and a filtrate.
  • the dosage form of the composition can be in a variety of dosage forms such as liquids, emulsions, suspensions, powders, granules, wettable powders or water-dispersible granules.
  • the Schizochytrium preparation may be a cracked algae powder.
  • Biomaterials preservation unit name China Microbial Culture Collection Management Committee General Microbiology Center
  • Screening plates A solid medium plate was prepared by pouring a screening solid medium of about 55 ° C into a Petri dish and cooling.
  • Fermentation medium 60 g of glucose, 10 g of glutamic acid or sodium glutamate, 10 g of corn syrup dry powder, 14 g of NaSO 4 , 0.5 g of KCl, 2.0 g of MgSO 4 , 1.0 g of K 2 SO 4 , 1.0 g of KH 2 PO 4 , (NH 4 ) 2 SO 4 1.0 g and CaCl 2 0.5 g were dissolved in 1 L of distilled water to adjust the pH to 6.0.
  • Wort agar medium 150 g of malt dipping powder is dissolved in 1 L of a mixed solution (mixed from 1 part by volume of natural sea water and 1 volume of distilled water), and the pH is natural; then agar powder is added to a concentration of 15 g/100 mL. Medium.
  • Natural mixed tocopherol is ADM company's product, the item number is MTS-90; rosemary is the product of Corinthian Trading (Shanghai) Co., Ltd. Guangzhou Branch, the product number is ROSEMARY 41-19-58; tea polyphenol is Fujian Likangyuan Bioengineering Co., Ltd., the product number is TP-98; erythorbic acid is the product of Zhengzhou Tuoyang Experimental Co., Ltd., the product number is ⁇ 98%; phytic acid is the product of Laiyang Wanjiwei Biological Engineering Co., Ltd.
  • the inventor of the present application collected a shredded pot from a plurality of mangroves in Yunxiao County, Zhangzhou City, Fujian province, and mixed to obtain a mixed solution; inoculated 0.5 mL of the mixed solution into 5 mL of screening liquid medium, and then cultured at 25 ° C, 200 rpm / min. 2d, the culture liquid was obtained.
  • the culture broth obtained in the step 1 was evenly spread on a selection plate, and statically cultured at 25 ° C for 2 days to produce a single colony.
  • step 3 single colonies were picked and inoculated into 5 mL of fermentation medium, and then cultured at 25 ° C, 200 rpm / min for 2 days to obtain a culture bacterial solution.
  • the culture liquid obtained in the step 3 was centrifuged at 4 ° C, 2000 rpm for 5 min, and the cells were collected.
  • the upper organic phase was placed in a glass weighing dish (which has been dried and weighed), and the glass was weighed. Place the dish on a boiling water bath in a fume hood to fully evaporate the organic phase (must be fully evaporated) and the liquid phase is grease.
  • step 6 Take the oil extracted in step 5, test the DHA content according to GB 26400-2011 national food safety standard, and test the composition and content of fatty acid according to the method of AOAC996.06.
  • the strain with a higher DHA content was selected and repeatedly purified 24 times.
  • One of the selected Schizochytrium strains was named as Schizontium HS01.
  • the Schizontium HS01 was inoculated into the fermentation medium for 12 passages and the DHA content was measured according to the above procedure. The results showed that the stability of DHA produced by Schizontium HS01 was good.
  • the Schizontium HS01 was inoculated onto the wort agar medium, and cultured at 25 ° C in the dark. After 5 days, the morphology of the colonies was observed and the morphological characteristics of the cells were observed by high-resolution transmission electron microscopy.
  • the Schizochytrium HS01 is a Schizoochytrium limacinum.
  • Schizoochytrium limacinum HS01 was deposited on March 10, 2017 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) The number is CGMCC No.13746.
  • the procedure for preparing the karst algae powder using the Schizochytrium sp. HS01 of Example 1 was as follows. The experiment was repeated three times, and the results were averaged:
  • the shake flask medium 1 is composed of a solute and a solvent, the solvent is water, the solute and its concentration are respectively 60 g/L glucose, and the yeast extract is 5 g/L;
  • the shake flask medium 2 is composed of a solute and a solvent, the solvent is water, the solute and The concentrations were 150 g/L glucose and yeast extract 25 g/L, respectively.
  • Seed medium 1 consists of solute and solvent.
  • the solvent is water.
  • the solute and its concentration are glucose 60g/L, yeast extract 8g/L, yeast powder 3g/L, Na 2 SO 4 5g/L, KCl 0.5g/L.
  • the seed medium 2 is composed of solute and solvent, the solvent is water, the solute and its concentration are respectively glucose 150g/L, yeast extract 25g/L, yeast powder 8g/L, Na 2 SO 4 20g/L, KCl 1.5g/L, MgSO 4 3.0g/L, K 2 SO
  • Fermentation medium 1 consists of solute and solvent.
  • the solvent is water.
  • the solute and its concentration are glucose 60g/L, yeast extract 8g/L, yeast powder 3g/L, Na 2 SO 4 5g/L, KCl 0.5g/L.
  • the solvent is water.
  • the solute and its concentration are glucose 150g/L, yeast paste 25g/L, yeast powder 8g/L, Na 2 SO 4 20g/L, KCl 1.5g/ L, MgSO 4 3.0 g/L, K 2 SO 4 2.5 g/L, KH 2 PO 4 2.0 g/L, (NH 4 ) 2 SO 4 5.0 g/L, CaCl 2 2.5 g/L, CuSO 4 0.02 g /L, ZnSO 4 0.02g / L, biotin 0.06g / L, sodium octenyl succinate 10g / L, whey protein powder 20g / L, after the completion of the use of alkali (sodium hydroxide solution or ammonia) Adjust the initial pH to 6.0.
  • alkali sodium hydroxide solution or ammonia
  • the Physalis algae HS01 was inoculated into the shake flask medium 1, and cultured at a rotation speed of 200 rpm and a temperature of 20 ° C for 24 hours to obtain a shake flask culture solution 1; the shake flask culture solution 1 was inoculated into the seed culture medium 1 to be dissolved.
  • Oxygen is 10 to 80% (dissolved oxygen is a dynamic process during growth), cultured at a temperature of 20 ° C for 48 h, and the pH of the culture solution is maintained between 4.5 and 6.5 during the cultivation, and the pH of the fermentation process is lowered.
  • the sodium hydroxide solution is adjusted to obtain the seed culture solution 1; the seed culture solution 1 is inoculated into the fermentation medium 1 according to the 10% inoculation amount, and the dissolved oxygen is 10 to 80% (the dissolved oxygen in the growth process is a dynamic
  • the process is carried out at a temperature of 20 ° C for 120 hours to obtain a fermentation broth.
  • the fermentation broth is recorded as the fermentation broth 1 , and the pH of the culture solution is maintained between 4.5 and 6.5 during the cultivation, and the pH of the fermentation process is lowered.
  • adjust with sodium hydroxide solution is adjusted to obtain the seed culture solution 1; the seed culture solution 1 is inoculated into the fermentation medium 1 according to the 10% inoculation amount, and the dissolved oxygen is 10 to 80% (the dissolved oxygen in the growth process is a dynamic
  • the process is carried out at a temperature of 20 ° C for 120 hours to obtain a fermentation broth.
  • the fermentation broth is recorded as the fermentation broth 1 , and the pH of the culture solution is maintained between 4.5 and
  • the Physalis algae HS01 was inoculated into the shake flask medium 2, and cultured at a rotation speed of 400 rpm and a temperature of 30 ° C for 48 hours to obtain a shake flask culture solution 2; the shake flask culture solution 2 was inoculated into the seed culture medium 2, and dissolved therein.
  • the culture is carried out for 24 hours under the condition of oxygen at 10 to 80% and temperature of 30 ° C.
  • the pH of the culture solution is maintained between 4.5 and 6.5 during the cultivation, and the pH of the fermentation process is lowered, and the pH is adjusted by ammonia or sodium hydroxide solution.
  • Seed culture solution 2 seed culture solution 2 was inoculated into fermentation medium 2 according to 20% inoculation amount, and cultured under the conditions of dissolved oxygen of 10 to 80% and temperature of 30 ° C for 72 hours to obtain a fermentation liquid, and the fermentation liquid was recorded.
  • the pH of the culture solution is maintained between 4.5 and 6.5 during the cultivation, and the pH of the fermentation process is lowered, and the pH is adjusted by ammonia water or sodium hydroxide solution.
  • the Physalis algae HS01 was inoculated into the shake flask medium 1, and cultured at a rotation speed of 200 rpm and a temperature of 20 ° C for 24 hours to obtain a shake flask culture solution 1; the shake flask culture solution 1 was inoculated into the seed culture medium 1 to be dissolved.
  • Oxygen is 10 ⁇ 80%, and the temperature is 20°C for 48h.
  • the pH of the culture solution is maintained between 4.5 and 6.5. The pH of the fermentation process will decrease and it will be adjusted by ammonia or sodium hydroxide solution.
  • Seed culture solution 1 seed culture solution 1 was inoculated into fermentation medium 3 at a seeding rate of 10%, and cultured under the conditions of 0 to 80% dissolved oxygen at a temperature of 20 ° C for 120 hours to obtain a fermentation liquid, and the fermentation liquid was recorded.
  • the pH of the culture solution is maintained between 4.5 and 6.5 during the cultivation, and the pH of the fermentation process is lowered, and the pH is adjusted by ammonia water or sodium hydroxide solution.
  • the Physalis algae HS01 was inoculated into the shake flask medium 2, and cultured under the conditions of a rotation speed of 400 rpm and a temperature of 30 ° C for 24 hours to obtain a shake flask culture solution 2; the shake flask culture solution 2 was inoculated to the seed culture medium 2, and dissolved therein.
  • Oxygen is 10 to 80%, and the temperature is 30 ° C for 24 h.
  • the pH of the culture solution is maintained between 4.5 and 6.5, the pH of the fermentation process is lowered, and the ammonia or water is adjusted to obtain Seed culture solution 2; seed culture solution 2 was inoculated into fermentation medium 4 according to the inoculation amount of 20%, and cultured under the conditions of dissolved oxygen of 10 to 80% and temperature of 30 ° C for 72 hours to obtain a fermentation liquid, and the fermentation liquid was recorded.
  • the pH of the culture solution is maintained between 4.5 and 6.5 during the cultivation, and the pH of the fermentation process is lowered, and the pH is adjusted by the ammonia water or the sodium hydroxide solution.
  • the antioxidant 1 is added to the fermentation liquid 1 (the antioxidant 1 is composed of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid, among which natural mixed tocopherol, rosemary, tea The mass ratio of polyphenols, isoascorbic acid and phytic acid is 20:2:10:10:2) to obtain a mixed liquid in which the quality of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid The percentage content is 0.2%, 0.02%, 0.1%, 0.1%, and 0.02%, respectively, and the mixture is emulsified and mixed to obtain a stable fermentation liquid 1; the stable fermentation liquid 1 is pasteurized, and then sprayed.
  • the cracked pot algae powder 1 was prepared by roller or freeze-drying.
  • antioxidant 2 consists of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid, among which natural mixed tocopherol, rosemary, tea polyphenol, and different
  • the mass ratio of ascorbic acid to phytic acid is 40:3:20:20:4
  • the mass percentage of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid in the mixture is respectively 0.4%, 0.03%, 0.2%, 0.2%, and 0.04%
  • the mixture is emulsified and mixed to obtain a stable fermentation liquid 2; the stable fermentation liquid 2 is pasteurized, and then sprayed, drum or
  • the cleavage algae powder 2 was obtained by freeze-drying.
  • the fermentation broth 3 is centrifuged to collect the cell mud of the phloem, and the same volume of sterile deionized water is added according to the volume of the granules of the phloem, and then centrifuged, and the washing is repeated 2 to 3 times to obtain the cell mud of the karyophylla; Adding antioxidant 3 to the cell salt of the karst algae (antioxidant 3 consists of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid, among which natural mixed tocopherol, rosemary, tea The mass ratio of phenol, isoascorbic acid and phytic acid is 60:2:40:30:6), and the mass percentage of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid in the mixture are respectively 0.6%, 0.02%, 0.4%, 0.3%, and 0.06%, the mixture is emulsified and mixed to obtain a stable cell
  • the fermentation broth 4 is centrifuged to collect the cell mud of the karyophylla, and the same volume of sterile deionized water is added according to the volume of the granules of the phloem, and then centrifuged, and the washing is repeated 2 to 3 times to obtain the cell mud of the karyophylla; Adding antioxidant 4 to the cell salt of the karst algae (antioxidant 4 consists of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid, among which natural mixed tocopherol, rosemary, tea The mass ratio of phenol, isoascorbic acid and phytic acid is 80:2:40:40:8), and the mass percentage of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid in the mixture are respectively 0.8%, 0.02%, 0.4%, 0.4%, and 0.08%, the mixture is emulsified and mixed to obtain a stable cell
  • the protein, moisture, fatty acid, ash and DHA content of the Phytophthora powder 1-4 obtained in the step 1 were respectively detected, and the specific detection methods are as follows:
  • Moisture detection According to GB 5009.3 "Food Safety National Standard for Determination of Moisture in Food".
  • the fatty acid composition was carried out according to GB 5009.168-2016 "Food Safety National Standard Food Determination of Fatty Acids" and GB/T 24894-2010/ISO 6800:1997 method.
  • the protein content of the cracked pot algae powder obtained in the above step is 10 to 60%, the water content is 0.5 to 3.0%, the mass content of the ash is 3 to 12%, and the mass content of the fatty acid is 25 to 50%.
  • the unsaturated fatty acid DHA has a mass content of 10 to 24%
  • the DPA has a mass content of 2.0 to 6.0%
  • the EPA has a mass content of 0.1 to 0.5%.
  • each indicator in Table 1 refers to the mass percentage of each substance in dry powder; other fatty acids refer to C8:0 octanoic acid, C10:0 acid, C16:0 palmitic acid, C18:0 stearic acid, C22: 5 DPA, C20: 5 EPA and C22: 6 fatty acids outside DHA.
  • the rumen fistula experiment of the cows with the cracked pot algae powders 1 and 2 was carried out by the nylon bag method. The steps are as follows:
  • a cow (Holstein cow) with a permanent gastric fistula.
  • deworming was carried out during this period, and the ectoparasite was removed by 1% trichlorfon solution, and the parasite was removed by oral administration of 0.8 mg/kg of levamisole hydrochloride.
  • the cracked pot algae powder was randomly collected by the "quadruple method", and dried at 65 ° C to a constant weight, and then placed in a grinding bottle for use.
  • a 300-mesh nylon cloth was cut into a rectangular shape of 170 mm ⁇ 130 mm, and after folding, a nylon bag having a size of 120 mm ⁇ 80 mm was formed by double-stitching with a polyester thread, and the loose side was flattened with a soldering iron. Before the test, put the nylon bag into the rumen balance for 72 hours, take it out and wash it, and check it without damage before use.
  • the rumen degradation of the nylon bag method was carried out according to the scheme proposed by the Dairy Cow Breeding Standard Scientific Research Cooperation Group. The test was designed using a random unit group, and each cow was given two repetitions at each time point.
  • Each nylon bag was filled with 10g of cracked pot algae powder, and the variance analysis of the sample was not significant (P>0.05).
  • Each 2 bags were tied to a 30 cm long semi-polyethylene tube.
  • the nylon bag was placed in the ventral sac of the rumen 2 hours after the morning feeding, and the other end of the tube was hung on the fistula cap.
  • Six tubes were placed in the rumen of each cow for a total of 12 bags. Take a tube from each rumen of each cow at 0h, 2h, 4h, 6h, 12h, 24h, 36h and 48h after bag release, wash with water, and rinse in the washing machine for 7 minutes. Clarify to water, then bake at 65 ° C to constant weight and weigh.
  • DM dry matter
  • the cracking pot algae powder of Example 3 and Example 2 can increase the DHA content in milk.
  • the cows were fed the cracked pot algae powder of Example 2, and the DHA content in the milk was measured to determine the effect of the cracked pot algae powder on the DHA content in the milk. The experiment was repeated three times.
  • the pre-feeding period of the cows in each experimental group was carried out (the pre-feeding period was 15 days, and the amount of cracked pot algae powder was gradually added according to the adaptation of the cows); after the formal feeding period, the cracked pot algae powder was added according to a certain proportion. Stir in the feed and mix and feed once every 8 hours.
  • the cracked pot algae powder added to the feed of the free-range experiment group 1 and the free-range experiment group 2 was the crack pot of the cracked pot algae powders 1 and 2 of Example 1, the captive experiment group 1 and the captive experiment group 2, respectively.
  • the algal flours were respectively the cracked pot algae powders 1 and 2 of Example 1, and the specific feeding methods were as follows:
  • Pre-feeding period The amount of algae powder fed to the experimental group is gradually increased, as follows:
  • the feeding amount of the cracked pot algae powder on the first and second days of the pre-feeding period was 50 mg/day/head;
  • the feeding amount of the cracked pot algae powder on the 3rd and 4th days of the pre-feeding period was 75 mg/day/head;
  • the feeding amount of the cracked pot algae powder on the 5th and 6th day of the pre-feeding period was 100 mg/day/head;
  • the feeding amount of the cracked pot algae powder on the 7th and 8th days of the pre-feeding period was 125 mg/day/head;
  • the feeding amount of the cracked pot algae powder on the 9th and 10th days of the pre-feeding period was 150 mg/day/head;
  • the feeding amount of the cracked pot algae powder on the 11th and 12th days of the pre-feeding period was 200 mg/day/head;
  • the feeding amount of the cracked pot algae powder on the 13th, 14th and 15th days of the pre-feeding period was 250 mg/day/head.
  • the formal feeding period is 250 mg/day/head of the cracked pot algae powder.
  • the feed of the cows was not added with the cracked pot algae powder, and the other components were the same as the experimental group.
  • the feeding time and feeding amount were the same as the experimental group.
  • the free-range experimental group 1, the free-range experimental group 2 and the free-range blank control group of the dairy cows were randomly reared, and there were no other edible foods for the dairy cows in the free-range field; the captive experimental group 1, the captive experimental group 2 and the captive blank control group of cows In captivity, there are no other edible foods in the circle.
  • DHA milk sample collection collect all the milk samples of cattle fed three times in the morning, evening, and mixed samples for inspection; and according to the national standard GB5413.27-2010 determination of fatty acids in infant food and dairy products; detect milk fat, milk protein, The DHA content index and the ratio of DH in the Sn-2 position in the milk to the total DHA.
  • the end of the pre-feeding period is the 15th day of the pre-feeding period
  • the 7th, 15th, 30th, 45th, 60th and 75th days of the official period are the 7th, 15th, 30th, 45th, 60th and 75th days of the official feeding period, respectively. After 75 days, it refers to the 100th day.
  • the cracked pot algae powder of Example 4 and Example 2 can improve egg quality and laying performance of laying hens
  • Example 2 the laying hen powder of Example 2 was fed to the laying hen to detect the effect of the cracked pot algae powder on laying performance and egg quality of the laying hen.
  • a total of 360 laying hens (Hailan white chicken) with no significant difference in healthy body weight were randomly selected. There was no significant difference in age between the laying hens.
  • the laying hens were randomly divided into 4 groups (control group, 0.5% experiment). Group, 1.0% experimental group and 1.5% experimental group), each group of 6 parallel, each parallel 15 .
  • the test diet was fed as a dry powder, fed three times a day, fed ad libitum and drinking water, and the daily feed intake was recorded by cage.
  • the basic diet of the laying hens was a corn-soybean meal type diet.
  • Each experimental group was fed with the basic diet supplemented with the cracked pot algae powder of Example 2, and the mass content of the cracked pot algae powder 1 in the 0.5% experimental group.
  • the mass content of the cracked pot algae powder was 1.0%, and 1.5% of the food in the experimental group was 1.5%; the control group was fed the basal diet.
  • the day when the experimental group was fed with the cracked pot algae powder was recorded as the first day.
  • the egg production rate was measured daily, and the cholesterol content of the eggs was measured on the 15th day and the 25th day respectively (the results on the 25th day are shown in Table 5), and the changes in egg production rate, egg cholesterol and DHA content were calculated. 6 is shown.
  • Table 5 Determination of egg production rate, cholesterol and DHA content on the 25th day of cultivating
  • “increased egg production rate” refers to the change in egg production rate of the laying hens in the same period of the control group.
  • “Cholesterol increase and decrease” refers to the cholesterol content of the eggs in the same period as the control group. The amount of change, "+” means increase, and “-” means decrease.
  • the egg production rate in the 0.5% group and the 1.5% group on the 25th day was not significantly different from the control group (P>0.05), but the 1.0% group could significantly improve the laying hens.
  • the egg production rate (P ⁇ 0.05) indicates that feeding a specific amount of schistosomiasis powder 1 can increase the laying rate of laying hens.
  • the laying hens were fed with the cracked pot algae powder 1.
  • the DHA content on the 15th day of feeding was higher than that of the control group, and the 0.5% group could increase by 317.11% to 150mg/100g; the 1.0% group could increase by 579.48% to 244mg/100g; and 1.5%.
  • the group can increase by 885.79% to 354 mg/100g.
  • the 0.5% and 1.0% groups remained essentially unchanged, while the 1.5% group showed a slight decrease, but there was still a significant increase compared to the control group. It is indicated that feeding the cracked pot algae powder 1 can increase the DHA content in the egg of the laying hen, and the more the amount of the cracked pot algae powder 1 in the food, the higher the DHA content in the egg.
  • the cracked pot algae powder 1 in the above step was replaced with the cracked pot algae powder 2, and the other steps were unchanged, and the same trend of change was obtained, indicating that the cracked pot algae powders 1 and 2 of Example 1 were both obtained. Has the same function.
  • the Schizochytrium algae powder prepared by Schizochytrium limacinum of the present invention can increase the content of DHA in animal products, can lower the cholesterol content in animal products, and can also improve the egg laying performance of poultry.
  • This natural source of high DHA content animal products, organic, safe, stable, easy to absorb, can be a safer and more effective way for people to ingest natural DHA, more able to cater to and meet consumer demand, and thus the Schizophrenia and the present invention
  • the cracked pot algae powder has a wide range of applications in the fields of general food and animal husbandry.

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Abstract

An application of schizochytrium limacinum and a preparation thereof in the improvement of the quality and yield of an animal product. The preservation number of schizochytrium limacinum in China General Microbiological Culture Collection Center is CGMCC No.13746. A method for improving the quality and yield of an animal product is implemented by feeding an animal with schizochytrium limacinum and the preparation thereof.

Description

裂壶藻及其制剂在提高动物产品品质及产量中的应用Application of Schizophyllum sp. and its preparation in improving animal product quality and yield 技术领域Technical field
本发明涉及农业生物中，裂壶藻及其制剂在提高动物产品品质及产量中的应用。The invention relates to the application of the genus Schizophyllum and its preparation in improving the quality and yield of animal products in agricultural organisms.
背景技术Background technique
DHA(二十二碳六烯酸)属于ω-3系列多不饱和脂肪酸(ω-3PUFAs)，是人体细胞膜和神经组织的重要组成物质，具有健脑益智、促进视神经的发育、防治老年痴呆等重要生理功能，在促进婴幼儿生长发育，预防心血管疾病，抑制和治疗某些癌症，保证神经***正常工作等方面起到重要作用。DHA (docosahexaenoic acid) belongs to the omega-3 series of polyunsaturated fatty acids (ω-3 PUFAs). It is an important component of human cell membrane and nerve tissue. It has brain-enhancing, promoting optic nerve development and preventing and treating senile dementia. Such important physiological functions play an important role in promoting the growth and development of infants and young children, preventing cardiovascular diseases, inhibiting and treating certain cancers, and ensuring the normal operation of the nervous system.
随着生活水平和消费水平的提高以及DHA生理功能研究的不断深入，对于不同人群的DHA摄入量问题越来越受到关注。根据中国食品协会完成的《中国人DHA食用情况调查》显示，中国大众每日从食物中直接DHA摄入量仅为约40mg，处于DHA严重“饥饿”状态。从饮食中获取DHA已成为人们的共识，其中以乳制品尤为普遍，富含高品质DHA的牛奶、羊奶等奶制品每年的需求量不断上升，从奶制品中摄取高品质DHA，已经成为一种趋势。With the improvement of living standards and consumption levels and the deepening of DHA physiological function research, more and more attention has been paid to the DHA intake problem of different populations. According to the "Chinese DHA Consumption Survey" completed by the China Food Association, the daily intake of DHA from the food by the Chinese public is only about 40mg, which is in a state of severe "hunger" in DHA. Obtaining DHA from the diet has become a consensus. Dairy products are especially popular. The annual demand for milk products such as milk and goat milk rich in high-quality DHA is increasing. The intake of high-quality DHA from dairy products has become one. Trends.
普通牛奶中的DHA含量极低，难以满足人们日常需求，而乳制品外源添加DHA则存在所需材料多，较大消耗企业生产成本，添加过程易造成DHA降价、分解或产生异味等问题。通过适当增加反刍动物日粮中的DHA等多不饱和脂肪酸摄入量，可提高乳汁、肌肉组织中DHA含量，但反刍动物特殊的消化结构使DHA等多不饱和脂肪酸在进入瘤胃后被大部分转化成饱和脂肪酸，极大降低了DHA等多不饱和脂肪酸利用率。市面上的脂肪酸过瘤胃保护技术主要有包被、氢化、钙化等，不仅处理技术难度大，生产成本很高，且存在日粮采食量降低、消化吸收率下降，产生乳脂下降综合征，甚至造成毒副作用等缺陷。因此，如何提高哺乳动物尤其是反刍动物乳汁中DHA的含量以及进一步利用反刍动物机体转化化生产富含DHA天然有机奶是目前急需解决的问题。The DHA content in ordinary milk is extremely low, which is difficult to meet people's daily needs. However, the addition of DHA to dairy products has more materials and consumes more production costs. The addition process is likely to cause DHA price reduction, decomposition or odor. By appropriately increasing the intake of polyunsaturated fatty acids such as DHA in ruminant diets, the DHA content in milk and muscle tissue can be increased, but the special digestive structure of ruminants makes most of the unsaturated fatty acids such as DHA enter the rumen and be mostly Conversion to saturated fatty acids greatly reduces the utilization of polyunsaturated fatty acids such as DHA. The fatty acid rumen protection technology on the market mainly includes coating, hydrogenation and calcification. It is not only difficult to handle, but also has high production cost, and there is a decrease in dietary feed intake, a decrease in digestion and absorption rate, and a creamy decline syndrome. Causes toxic side effects and other defects. Therefore, how to increase the content of DHA in the milk of mammals, especially ruminants, and further utilize the transformation of ruminant organisms to produce DHA-rich natural organic milk is an urgent problem to be solved.
磷脂酰丝氨酸(phosphatidylserine，PS)是一种天然的磷脂，PS的结构决定了其具有双亲性的独特性质，带有负电荷的一端具有亲水性(或水溶性)，由脂肪酸组成的另一端具有亲脂性(或脂溶性)。研究表明，PS可以作为DHA的一种载体，当DHA结合到磷脂酰丝氨酸甘油骨架2位时，DHA的稳定性更高，而且更易通过血脑屏障。DHA和PS在体外以2-DHA-PS(即Sn-2位DHA)的形式被吸收后，在大脑中最终转化为DHA-PS而进行神经保护作用，2-DHA-PS可以兼具DHA和PS的生物学功能。因此，如何提高鸡蛋中DHA的含量，以丰富不同型态DHA的来源，扩展DHA的应用领域和消费范围，是一个需要解决的问题。Phosphatidylserine (PS) is a natural phospholipid. The structure of PS determines its unique amphiphilic nature. The negatively charged end is hydrophilic (or water soluble) and the other end is composed of fatty acids. It is lipophilic (or fat soluble). Studies have shown that PS can be used as a carrier of DHA. When DHA binds to the 2 position of phosphatidylserine glycerol backbone, DHA is more stable and more easily passes through the blood-brain barrier. DHA and PS are absorbed in the form of 2-DHA-PS (ie, Sn-2 position DHA) in vitro, and finally converted into DHA-PS in the brain for neuroprotection. 2-DHA-PS can have both DHA and The biological function of PS. Therefore, how to increase the content of DHA in eggs, enrich the source of different types of DHA, and expand the application field and consumption range of DHA is a problem to be solved.
发明公开Invention disclosure
本发明所要解决的技术问题是如何提高动物产品品质和产量。The technical problem to be solved by the present invention is how to improve the quality and yield of animal products.
为解决上述技术问题，本发明首先提供了裂壶藻(Schizochytrium  limacinum)或其制剂的下述任一应用：In order to solve the above technical problems, the present invention first provides any of the following applications of Schizochytrium limacinum or a preparation thereof:
A1)在提高动物产品品质中的应用；A1) Application in improving the quality of animal products;
A2)在制备提高动物产品品质的物质中的应用；A2) application in the preparation of a substance for improving the quality of an animal product;
A3)在提高动物产品产量中的应用；A3) Application in increasing the yield of animal products;
A4)在制备提高动物产品产量的物质中的应用。A4) Use in the preparation of a substance which increases the yield of animal products.
上述应用中，所述裂壶藻(Schizochytrium limacinum)可为裂壶藻(Schizochytrium limacinum)HS01，所述裂壶藻(Schizochytrium limacinum)HS01在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.13746。In the above application, the Schizochytrium limacinum may be Schizochytrium limacinum HS01, and the preservation number of the Schizochytrium limacinum HS01 in the General Microbiology Center of the Chinese Collection of Microorganisms and Cultures is CGMCC. No. 13746.
所述制剂的活性成分可为所述裂壶藻(Schizochytrium limacinum)。The active ingredient of the formulation may be the Schizochytrium limacinum.
上述应用中，所述提高动物产品品质可为提高所述动物产品DHA含量和/或提高所述动物产品Sn-2位DHA含量和/或降低所述动物产品胆固醇含量。In the above application, the improving the quality of the animal product may be to increase the DHA content of the animal product and/or increase the DHA content of the animal product at the Sn-2 position and/or reduce the cholesterol content of the animal product.
上述应用中，所述制剂可为裂壶藻粉。In the above application, the preparation may be a cracked algae powder.
上述应用中，所述制剂可按照包括如下步骤的方法(将该方法记为裂壶藻制剂的制备方法)制备：培养所述裂壶藻(Schizochytrium limacinum)，得到发酵液；利用所述发酵液制备得到所述制剂。In the above application, the preparation may be prepared according to a method comprising the following steps (the method is referred to as a preparation method of a Schizochytrium preparation): cultivating the Schizochytrium limacinum to obtain a fermentation broth; using the fermentation broth The formulation was prepared.
上述应用中，培养所述裂壶藻(Schizochytrium limacinum)可利用发酵培养基进行，所述发酵培养基由溶剂和溶质组成，所述溶剂为水，所述溶质及其浓度分别为葡萄糖60～150g/L、酵母膏8～25g/L、酵母粉3～8g/L、Na 2SO 4 5～20g/L、KCl 0.5～1.5g/L、MgSO 4 1.0～3.0g/L、K 2SO 4 0.5～2.5g/L、KH 2PO 4 1.0～2.0g/L、(NH 4) 2SO 4 2.0～5.0g/L、CaCl 2 0.5～2.5g/L、CuSO 4 0.001～0.02g/L、ZnSO 4 0.001～0.02g/L、生物素0.001～0.06g/L、淀粉0.1～10g/L和蛋白粉0～20g/L，pH为4.5～6.5。 In the above application, the culture of the Schizochytrium limacinum can be carried out using a fermentation medium consisting of a solvent and a solute, the solvent being water, the solute and its concentration being glucose 60-150 g, respectively. /L, yeast extract 8 ~ 25g / L, yeast powder 3 ~ 8g / L, Na 2 SO 4 5 ~ 20g / L, KCl 0.5 ~ 1.5g / L, MgSO 4 1.0 ~ 3.0g / L, K 2 SO 4 0.5 to 2.5 g/L, KH 2 PO 4 1.0 to 2.0 g/L, (NH 4 ) 2 SO 4 2.0 to 5.0 g/L, CaCl 2 0.5 to 2.5 g/L, CuSO 4 0.001 to 0.02 g/L, ZnSO 4 is 0.001 to 0.02 g/L, biotin is 0.001 to 0.06 g/L, starch is 0.1 to 10 g/L, and protein powder is 0 to 20 g/L, and the pH is 4.5 to 6.5.
所述淀粉可为玉米淀粉或辛烯基琥珀酸淀粉钠，所述蛋白粉可为豌豆蛋白粉或乳清蛋白粉。所述发酵培养基的pH具体可为6。The starch may be corn starch or sodium starch octenyl succinate, and the protein powder may be pea protein powder or whey protein powder. The pH of the fermentation medium may specifically be 6.
豌豆蛋白粉是从豌豆中提取得到的豌豆总蛋白质。Pea protein powder is the total protein of pea extracted from peas.
乳清蛋白粉是从牛奶中提取得到的牛奶总蛋白质。Whey protein powder is the total protein of milk extracted from milk.
所述发酵培养基的溶质及其浓度具体可为如下n1)或n2)或n3)或n4)：The solute of the fermentation medium and its concentration may specifically be as follows: n1) or n2) or n3) or n4):
n1)葡萄糖60g/L、酵母膏8g/L、酵母粉3g/L、Na 2SO 4 5g/L、KCl 0.5g/L、MgSO 4 1.0g/L、K 2SO 4 0.5g/L、KH 2PO 4 1.0g/L、(NH 4) 2SO 4 2.0g/L、CaCl 2 0.5g/L、CuSO 4 0.001g/L、ZnSO 4 0.001g/L、生物素0.001g/L和玉米淀粉0.1g/L； N1) glucose 60g/L, yeast extract 8g/L, yeast powder 3g/L, Na 2 SO 4 5g/L, KCl 0.5g/L, MgSO 4 1.0g/L, K 2 SO 4 0.5g/L, KH 2 PO 4 1.0g/L, (NH 4 ) 2 SO 4 2.0g/L, CaCl 2 0.5g/L, CuSO 4 0.001g/L, ZnSO 4 0.001g/L, biotin 0.001g/L and corn starch 0.1g/L;
n2)葡萄糖150g/L、酵母膏25g/L、酵母粉8g/L、Na 2SO 4 20g/L、KCl 1.5g/L、MgSO 4 3.0g/L、K 2SO 4 2.5g/L、KH 2PO 4 2.0g/L、(NH 4) 2SO 4 5.0g/L、CaCl 2 2.5g/L、CuSO 4 0.02g/L、ZnSO 4 0.02g/L、生物素0.06g/L、玉米淀粉10g/L和豌豆蛋白粉20g/L； N2) glucose 150g/L, yeast extract 25g/L, yeast powder 8g/L, Na 2 SO 4 20g/L, KCl 1.5g/L, MgSO 4 3.0g/L, K 2 SO 4 2.5g/L, KH 2 PO 4 2.0g/L, (NH 4 ) 2 SO 4 5.0g/L, CaCl 2 2.5g/L, CuSO 4 0.02g/L, ZnSO 4 0.02g/L, biotin 0.06g/L, corn starch 10g / L and pea protein powder 20g / L;
n3)葡萄糖60g/L、酵母膏8g/L、酵母粉3g/L、Na 2SO 4 5g/L、KCl 0.5 g/L、MgSO 4 1.0g/L、K 2SO 4 0.5g/L、KH 2PO 4 1.0g/L、(NH 4) 2SO 4 2.0g/L、CaCl 2 0.5g/L、CuSO 4 0.001g/L、ZnSO 4 0.001g/L、生物素0.001g/L和辛烯基琥珀酸淀粉钠0.1g/L； N3) glucose 60g/L, yeast extract 8g/L, yeast powder 3g/L, Na 2 SO 4 5g/L, KCl 0.5 g/L, MgSO 4 1.0g/L, K 2 SO 4 0.5g/L, KH 2 PO 4 1.0g/L, (NH 4 ) 2 SO 4 2.0g/L, CaCl 2 0.5g/L, CuSO 4 0.001g/L, ZnSO 4 0.001g/L, biotin 0.001g/L and octene Sodium succinate sodium starch 0.1g / L;
n4)葡萄糖150g/L、酵母膏25g/L、酵母粉8g/L、Na 2SO 4 20g/L、KCl 1.5g/L、MgSO 4 3.0g/L、K 2SO 4 2.5g/L、KH 2PO 4 2.0g/L、(NH 4) 2SO 4 5.0g/L、CaCl 2 2.5g/L、CuSO 4 0.02g/L、ZnSO 4 0.02g/L、生物素0.06g/L、辛烯基琥珀酸淀粉钠10g/L和乳清蛋白粉20g/L。 N4) glucose 150g/L, yeast extract 25g/L, yeast powder 8g/L, Na 2 SO 4 20g/L, KCl 1.5g/L, MgSO 4 3.0g/L, K 2 SO 4 2.5g/L, KH 2 PO 4 2.0g/L, (NH 4 ) 2 SO 4 5.0g/L, CaCl 2 2.5g/L, CuSO 4 0.02g/L, ZnSO 4 0.02g/L, biotin 0.06g/L, octene Sodium starch succinate 10 g / L and whey protein powder 20 g / L.
上述应用中，所述利用所述发酵液制备得到所述制剂可包括：干燥所述发酵液，得到所述制剂。In the above application, the preparing the preparation by using the fermentation liquid may include: drying the fermentation liquid to obtain the preparation.
上述方法还可包括在得到所述发酵液后向所述发酵液中添加抗氧化剂后再进行干燥得到所述裂壶藻粉(即所述制剂)。The above method may further comprise adding an antioxidant to the fermentation broth after obtaining the fermentation broth, and then drying to obtain the cleavage algae powder (ie, the preparation).
所述抗氧化剂可为油溶抗氧化剂和/或水溶抗氧化剂。所述油溶抗氧化剂可为迷迭香、天然混合生育酚、茶多酚和/或抗坏血酸棕榈酸酯。所述水溶抗氧化剂可为植酸、抗坏血酸和/或异抗坏血酸。The antioxidant can be an oil soluble antioxidant and/or a water soluble antioxidant. The oil soluble antioxidant may be rosemary, natural mixed tocopherol, tea polyphenols and/or ascorbyl palmitate. The water soluble antioxidant can be phytic acid, ascorbic acid and/or isoascorbic acid.
所述抗氧化剂在由几种不同的具体抗氧化剂组成时，各成分间的配比无要求，可根据具体需要调整。When the antioxidant is composed of several different specific antioxidants, the ratio between the components is not required, and can be adjusted according to specific needs.
所述抗氧化剂具体可为由天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸组成的混合抗氧化剂。所述混合抗氧化剂中各物质间的配比可为下述p1)、p2)、p3)或p4)：The antioxidant may specifically be a mixed antioxidant composed of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid. The ratio between the substances in the mixed antioxidant may be the following p1), p2), p3) or p4):
p1)天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸的质量比为20:2:10:10:2；P1) mass ratio of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid is 20:2:10:10:2;
p2)天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸的质量比为40:3:20:20:4；P2) the mass ratio of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid is 40:3:20:20:4;
p3)天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸的质量比为60:2:40:30:6；P3) the mass ratio of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid is 60:2:40:30:6;
p4)天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸的质量比为80:2:40:40:8。P4) The mass ratio of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid is 80:2:40:40:8.
所述干燥可为喷雾干燥或滚筒干燥或冷冻干燥。The drying can be spray drying or drum drying or freeze drying.
上述方法还可包括将所述发酵液中的所述裂壶藻(Schizochytrium limacinum)进行洗涤。上述方法进一步还可包括向洗涤后的裂壶藻(Schizochytrium limacinum)中添加所述抗氧化剂后再进行干燥。The above method may further comprise washing the Schizochytrium limacinum in the fermentation broth. The above method may further comprise adding the antioxidant to the washed Schizochytrium limacinum and then drying.
所述培养的溶氧量可为0～80％(如10～80％)。所述培养的温度可为20～30℃。所述培养的时间可为72～120h。The amount of dissolved oxygen in the culture may be 0 to 80% (e.g., 10 to 80%). The temperature of the culture may be 20 to 30 °C. The culture time may be 72 to 120 hours.
上述应用中，所述动物可为a1)或a2)或a3)：In the above application, the animal may be a1) or a2) or a3):
a1)家禽；A1) poultry;
a2)鸡；A2) chicken;
a3)北京白鸡、海兰白鸡、海兰褐蛋鸡或海兰粉壳鸡。A3) Beijing White Chicken, Hailan White Chicken, Hailan Brown Layer Chicken or Hailan Powder Shell Chicken.
所述动物产品可为所述动物产的蛋。The animal product can be an egg produced by the animal.
上述应用中，所述动物可为b1)或b2)：In the above application, the animal may be b1) or b2):
b1)反刍动物；B1) ruminants;
b1)牛。B1) cattle.
所述牛可为奶牛。所述奶牛可为荷斯坦奶牛。The cow can be a cow. The cow may be a Holstein cow.
所述动物产品可为所述动物的乳汁，如牛奶。The animal product can be the milk of the animal, such as milk.
所述裂壶藻制剂的制备方法也属于本发明的保护范围。The preparation method of the Schizochytrium preparation is also within the scope of protection of the present invention.
下述任一产品也属于本发明的保护范围：Any of the following products is also within the scope of protection of the present invention:
Y1)用于培养所述裂壶藻(Schizochytrium limacinum)的培养基，为所述发酵培养基；Y1) a medium for cultivating the Schizochytrium limacinum, which is the fermentation medium;
Y2)所述制剂。Y2) the formulation.
为解决上述技术问题，本发明还提供了提高动物产品品质的方法，所述方法包括：饲喂动物裂壶藻(Schizochytrium limacinum)或其制剂实现提高所述动物产品品质。In order to solve the above technical problems, the present invention also provides a method for improving the quality of an animal product, which comprises: feeding an animal Schizochytrium limacinum or a preparation thereof to improve the quality of the animal product.
所述裂壶藻(Schizochytrium limacinum)可为所述裂壶藻(Schizochytrium limacinum)HS01。The Schizochytrium limacinum may be the Schizochytrium limacinum HS01.
所述制剂的活性成分可为所述裂壶藻(Schizochytrium limacinum)。The active ingredient of the formulation may be the Schizochytrium limacinum.
上述方法中，所述提高动物产品品质可为c1)和/或c2)和/或c3)：In the above method, the improving animal product quality may be c1) and/or c2) and/or c3):
c1)提高所述动物产品DHA含量；C1) increasing the DHA content of the animal product;
c2)提高所述动物产品Sn-2位DHA含量；C2) increasing the DHA content of the animal product at the Sn-2 position;
c3)降低所述动物产品胆固醇含量。C3) reducing the cholesterol content of the animal product.
上述方法中，所述动物可为反刍动物，所述动物的所述裂壶藻(Schizochytrium limacinum)或其制剂饲喂量可为d1)-d7)中的任一种：In the above method, the animal may be a ruminant, and the Schizochytrium limacinum or the preparation thereof may be fed in any one of d1)-d7):
d1)50-500mg/天/头；D1) 50-500 mg / day / head;
d2)50-250mg/天/头；D2) 50-250 mg / day / head;
d3)75-250mg/天/头；D3) 75-250 mg / day / head;
d4)100-250mg/天/头；D4) 100-250mg / day / head;
d5)125-250mg/天/头；D5) 125-250 mg / day / head;
d6)150-250mg/天/头；D6) 150-250mg / day / head;
d7)200-250mg/天/头。D7) 200-250 mg / day / head.
所述动物可为家禽，饲喂所述动物的食物中所述裂壶藻(Schizochytrium limacinum)或其制剂的质量含量可为e1)-e3)中的任一种：The animal may be a poultry, and the mass of the Schizochytrium limacinum or the preparation thereof may be any one of e1)-e3) in the food to which the animal is fed:
e1)0.5％-2.5％；E1) 0.5% - 2.5%;
e2)0.5％-1.5％；E2) 0.5%-1.5%;
e3)1％-1.5％。E3) 1% - 1.5%.
所述动物为家禽，所述动物的食物由基础日粮与所述裂壶藻(Schizochytrium limacinum)或其制剂组成。所述基础日粮可为玉米-豆粕型 日粮。The animal is a poultry whose food consists of a basal diet and the Schizochytrium limacinum or a preparation thereof. The basal diet may be a corn-soybean meal type diet.
上述方法中，所述反刍动物可为牛。In the above method, the ruminant may be a cow.
所述牛可为奶牛。所述奶牛可为荷斯坦奶牛。The cow can be a cow. The cow may be a Holstein cow.
所述动物产品可为所述动物的乳汁，如牛奶。The animal product can be the milk of the animal, such as milk.
上述方法中，所述家禽可为a2)或a3)：In the above method, the poultry may be a2) or a3):
a2)鸡；A2) chicken;
a3)北京白鸡、海兰白鸡、海兰褐蛋鸡或海兰粉壳鸡。A3) Beijing White Chicken, Hailan White Chicken, Hailan Brown Layer Chicken or Hailan Powder Shell Chicken.
所述动物产品可为所述动物产的蛋。The animal product can be an egg produced by the animal.
上述方法中，所述制剂可利用所述裂壶藻制剂的制备方法制备。In the above method, the preparation can be prepared by the preparation method of the cleavage algae preparation.
利用所述提高动物产品品质的方法制备得到的动物产品或对所述动物产品加工得到的产品，也属于本发明的保护范围。It is also within the scope of the present invention to prepare an animal product prepared by the method for improving the quality of an animal product or a product obtained by processing the animal product.
上述产品中，所述动物为b1)或b2)：In the above products, the animal is b1) or b2):
b1)反刍动物；B1) ruminants;
b1)牛；B1) cattle;
所述动物产品为奶。The animal product is milk.
上述产品中，所述对所述动物产品加工得到的产品为f1)-f6)中的任一种：In the above product, the product obtained by processing the animal product is any one of f1)-f6):
f1)原生易吸收的DHA乳制品；F1) natively absorbable DHA dairy products;
f2)原生DHA纯牛奶F2) Native DHA pure milk
f3)原生DHA巴氏奶F3) Native DHA pasteurized milk
f4)原生DHA酸奶F4) Native DHA yogurt
f5)原生DHA奶粉F5) Native DHA milk powder
f6)酸奶。F6) Yogurt.
上述产品中，所述动物为a1)或a2)或a3)：In the above products, the animal is a1) or a2) or a3):
a1)家禽；A1) poultry;
a2)鸡；A2) chicken;
a3)北京白鸡、海兰白鸡、海兰褐蛋鸡或海兰粉壳鸡；A3) Beijing white chicken, Hailan white chicken, Hailan brown laying hen or Hailan powder shell chicken;
所述动物产品为蛋。The animal product is an egg.
上述产品中，所述对所述动物产品加工得到的产品为原生磷脂型DHA蛋制品。In the above product, the product obtained by processing the animal product is a native phospholipid DHA egg product.
为解决上述技术问题，本发明还提供了提高动物产品产量的方法，所述方法包括：饲喂动物裂壶藻(Schizochytrium limacinum)或其制剂实现提高所述动物产品产量。In order to solve the above technical problems, the present invention also provides a method for increasing the yield of an animal product, the method comprising: feeding an animal Schizochytrium limacinum or a preparation thereof to increase the yield of the animal product.
所述裂壶藻(Schizochytrium limacinum)可为所述裂壶藻(Schizochytrium limacinum)HS01。The Schizochytrium limacinum may be the Schizochytrium limacinum HS01.
所述制剂的活性成分可为所述裂壶藻(Schizochytrium limacinum)。The active ingredient of the formulation may be the Schizochytrium limacinum.
所述动物产品可为所述动物产的蛋。The animal product can be an egg produced by the animal.
上述方法中，所述制剂可利用所述裂壶藻制剂的制备方法制备。In the above method, the preparation can be prepared by the preparation method of the cleavage algae preparation.
上述方法中，所述动物为家禽，饲喂所述动物的食物中所述裂壶藻(Schizochytrium limacinum)或其制剂的质量含量为e1)-e3)中的任一种：In the above method, the animal is poultry, and the food of the animal is fed with any one of the mass content of the Schizochytrium limacinum or the preparation thereof, e1)-e3):
e1)0.5％-5％；E1) 0.5%-5%;
e2)0.5％-1.5％；E2) 0.5%-1.5%;
e3)1％-1.5％。E3) 1% - 1.5%.
上述方法中，所述家禽可为a2)或a3)：In the above method, the poultry may be a2) or a3):
a2)鸡；A2) chicken;
a3)北京白鸡、海兰白鸡、海兰褐蛋鸡或海兰粉壳鸡。A3) Beijing White Chicken, Hailan White Chicken, Hailan Brown Layer Chicken or Hailan Powder Shell Chicken.
本发明中，所述制剂还可以包括载体。所述载体可为固体载体或液体载体。所述固体载体可为矿物材料、植物材料或高分子化合物；所述矿物材料可为粘土、滑石、高岭土、蒙脱石、白碳、沸石、硅石和硅藻土中的至少一种；所述植物材料可为玉米粉、豆粉和淀粉中的至少一种；所述高分子化合物可为聚乙烯醇和/或聚二醇。所述液体载体可为有机溶剂、植物油、矿物油或水；所述有机溶剂可为癸烷和/或十二烷。所述菌剂中，所述活性成分可以以被培养的活细胞、活细胞的发酵液、细胞培养物的滤液或细胞与滤液的混合物的形式存在。所述组合物的剂型可为多种剂型，如液剂、乳剂、悬浮剂、粉剂、颗粒剂、可湿性粉剂或水分散粒剂。具体的，所述裂壶藻制剂可为裂壶藻粉。In the present invention, the preparation may further include a carrier. The carrier can be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; The plant material may be at least one of corn flour, soy flour, and starch; the polymer compound may be polyvinyl alcohol and/or polyglycol. The liquid carrier can be an organic solvent, vegetable oil, mineral oil or water; the organic solvent can be decane and/or dodecane. In the microbial agent, the active ingredient may be present as a cultured living cell, a fermentation broth of a living cell, a filtrate of a cell culture, or a mixture of a cell and a filtrate. The dosage form of the composition can be in a variety of dosage forms such as liquids, emulsions, suspensions, powders, granules, wettable powders or water-dispersible granules. Specifically, the Schizochytrium preparation may be a cracked algae powder.
生物材料保藏说明Biomaterial preservation instructions
生物材料的分类命名：Schizochytrium limacinumClassification of biological materials: Schizochytrium limacinum
生物材料的菌株编号：HS01Strain number of biomaterial: HS01
生物材料的保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心Biomaterials preservation unit name: China Microbial Culture Collection Management Committee General Microbiology Center
生物材料的保藏单位简称：CGMCCBiomaterials depositor referred to as: CGMCC
生物材料的保藏单位地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮政编码：100101Biomaterials Depository Address: No. 3, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101
生物材料的保藏日期：2017年3月10日Date of preservation of biological materials: March 10, 2017
生物材料的保藏中心登记入册编号：CGMCC No.13746Collection of Biomaterials Registration Number: CGMCC No.13746
实施发明的最佳方式The best way to implement the invention
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。The present invention is further described in detail with reference to the preferred embodiments thereof. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. For the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged.
筛选液体培养基：将葡萄糖50g和酵母粉15g溶于1L混合液(由1体积份天然海水和1体积蒸馏水混合而成)，pH值自然。Screening of liquid medium: 50 g of glucose and 15 g of yeast powder were dissolved in a 1 L mixture (mixed from 1 part by volume of natural sea water and 1 volume of distilled water), and the pH was natural.
筛选平板：将约55℃的筛选固体培养基倒入培养皿，冷却后得到的固体平板。Screening plates: A solid medium plate was prepared by pouring a screening solid medium of about 55 ° C into a Petri dish and cooling.
发酵培养基：将葡萄糖60g、谷氨酸或谷氨酸钠10g、玉米浆干粉10g、NaSO 4 14g、KCl 0.5g、MgSO 4 2.0g、K 2SO 4 1.0g、KH 2PO 4 1.0g、(NH 4) 2SO 4 1.0g和CaCl 20.5g溶于1L蒸馏水，调节pH值至6.0。 Fermentation medium: 60 g of glucose, 10 g of glutamic acid or sodium glutamate, 10 g of corn syrup dry powder, 14 g of NaSO 4 , 0.5 g of KCl, 2.0 g of MgSO 4 , 1.0 g of K 2 SO 4 , 1.0 g of KH 2 PO 4 , (NH 4 ) 2 SO 4 1.0 g and CaCl 2 0.5 g were dissolved in 1 L of distilled water to adjust the pH to 6.0.
麦芽汁琼脂培养基：将150g麦芽浸粉溶于1L混合液(由1体积份天然海水和1体积蒸馏水混合而成)，pH值自然；然后加入琼脂粉至其浓度为15g/100mL，得到的培养基。Wort agar medium: 150 g of malt dipping powder is dissolved in 1 L of a mixed solution (mixed from 1 part by volume of natural sea water and 1 volume of distilled water), and the pH is natural; then agar powder is added to a concentration of 15 g/100 mL. Medium.
天然混合生育酚为ADM公司产品，货号为MTS-90；迷迭香为科耐欧贸易(上海)有限公司广州分公司公司产品，货号为ROSEMARY 41-19-58；茶多酚为福建利康源生物工程有限公司产品，货号为TP-98；异抗坏血酸为郑州拓洋实验有限公司产品，货号为含量≥98％；植酸为莱阳市万基威生物工程有限公司产品。Natural mixed tocopherol is ADM company's product, the item number is MTS-90; rosemary is the product of Corinthian Trading (Shanghai) Co., Ltd. Guangzhou Branch, the product number is ROSEMARY 41-19-58; tea polyphenol is Fujian Likangyuan Bioengineering Co., Ltd., the product number is TP-98; erythorbic acid is the product of Zhengzhou Tuoyang Experimental Co., Ltd., the product number is ≥98%; phytic acid is the product of Laiyang Wanjiwei Biological Engineering Co., Ltd.
实施例1、裂殖壶HS01的分离鉴定Example 1. Isolation and identification of Schizontium HS01
一、裂殖壶HS01的分离First, the separation of the fission kettle HS01
1、本申请的发明人从福建省漳州市云霄县红树林多处采集裂殖壶，混合，得到混合液；将0.5mL混合液接种至5mL筛选液体培养基，然后25℃、200rpm/min培养2d，得到培养菌液。1. The inventor of the present application collected a shredded pot from a plurality of mangroves in Yunxiao County, Zhangzhou City, Fujian Province, and mixed to obtain a mixed solution; inoculated 0.5 mL of the mixed solution into 5 mL of screening liquid medium, and then cultured at 25 ° C, 200 rpm / min. 2d, the culture liquid was obtained.
2、将步骤1得到的培养菌液均匀涂布于筛选平板上，25℃静置培养2d，产生单菌落。2. The culture broth obtained in the step 1 was evenly spread on a selection plate, and statically cultured at 25 ° C for 2 days to produce a single colony.
3、完成步骤2后，分别挑取单菌落接种至5mL发酵培养基，然后25℃、200rpm/min培养2d，得到培养菌液。3. After completing step 2, single colonies were picked and inoculated into 5 mL of fermentation medium, and then cultured at 25 ° C, 200 rpm / min for 2 days to obtain a culture bacterial solution.
4、取步骤3得到的培养菌液，4℃、2000rpm离心5min，收集菌体。4. The culture liquid obtained in the step 3 was centrifuged at 4 ° C, 2000 rpm for 5 min, and the cells were collected.
5、取菌体1.0～2.0g至具塞量筒(规格为100mL)中，先加入15mL浓度为8.3mol/L的HCl水溶液，盖好盖子，置于70～80℃水浴中水解50～60min(期间每10min置于漩涡混合器上振荡具塞量筒1次)；待冷却到室温后，先加入10mL 95％(v/v)乙醇水溶液，充分振摇均匀后，再加入20mL无水***充分振摇萃取1～2min，最后加入20mL石油醚，充分振摇萃取1～2min，静置分层，将上层有机相置于玻璃称量皿(已烘干称过空重)，将该玻璃称量皿置于通风橱中的沸水浴上使有机相充分蒸发干净(务必充分挥发干净)，液相即为油脂。5, take the bacteria 1.0 ~ 2.0g into a measuring cylinder (specification of 100mL), first add 15mL of 8.3mol / L HCl aqueous solution, cover the lid, placed in a 70 ~ 80 ° C water bath for 50 ~ 60min hydrolysis ( During the 10 min period, it was placed on a vortex mixer and shaken with a measuring cylinder once); after cooling to room temperature, first add 10 mL of 95% (v/v) ethanol aqueous solution, shake well, then add 20 mL of anhydrous ether to fully vibrate. The mixture was shaken for 1~2 min, and finally 20 mL of petroleum ether was added. The mixture was shaken for 1 to 2 min, and the layer was allowed to stand. The upper organic phase was placed in a glass weighing dish (which has been dried and weighed), and the glass was weighed. Place the dish on a boiling water bath in a fume hood to fully evaporate the organic phase (must be fully evaporated) and the liquid phase is grease.
6、取步骤5提取的油脂，按照GB 26400-2011食品安全国家标准检测DHA含量，按照AOAC996.06的方法检测脂肪酸的组成及含量。6. Take the oil extracted in step 5, test the DHA content according to GB 26400-2011 national food safety standard, and test the composition and content of fatty acid according to the method of AOAC996.06.
挑选DHA含量较高的菌株，反复纯化24次。将筛选到的一株裂殖壶菌株命名为裂殖壶HS01。The strain with a higher DHA content was selected and repeatedly purified 24 times. One of the selected Schizochytrium strains was named as Schizontium HS01.
将裂殖壶HS01单克隆接种至发酵培养基连续传代12代并按照上述步骤检测DHA含量。结果表明，裂殖壶HS01生产DHA的稳定性良好。The Schizontium HS01 was inoculated into the fermentation medium for 12 passages and the DHA content was measured according to the above procedure. The results showed that the stability of DHA produced by Schizontium HS01 was good.
二、裂殖壶HS01的鉴定Second, the identification of the fission kettle HS01
1、形态学鉴定1. Morphological identification
将裂殖壶HS01接种至麦芽汁琼脂培养基上，25℃暗培养，5d后观察菌落的 形态并通过高分辨率透射电镜分析观察菌体的形态特征。The Schizontium HS01 was inoculated onto the wort agar medium, and cultured at 25 ° C in the dark. After 5 days, the morphology of the colonies was observed and the morphological characteristics of the cells were observed by high-resolution transmission electron microscopy.
结果表明，裂殖壶HS01的菌落直径为2～4.3mm，白色(后期浅橙色)，边缘不整齐；菌体以裂殖方式进行增殖，细胞壁薄，球形，无色或浅橙色，透明，大小为4.5～15.5μm，游动孢子及外质网未见。The results showed that the colony diameter of Schizontium HS01 was 2～4.3mm, white (late orange) and the edges were not neat. The cells proliferated by fission, the cell wall was thin, spherical, colorless or light orange, transparent and large. For 4.5 to 15.5 μm, no visible zoospores and exogenous nets were observed.
2、18s rDNA序列同源性分析2, 18s rDNA sequence homology analysis
裂殖壶HS01的18s rDNA的部分序列如序列表中的序列1所示。The partial sequence of the 18s rDNA of the Schizochyphora HS01 is shown as Sequence 1 in the Sequence Listing.
裂殖壶HS01的18s rDNA的部分序列如序列表中的序列2所示。The partial sequence of the 18s rDNA of the Schizochyphora HS01 is shown in SEQ ID NO: 2 in the Sequence Listing.
综合上述各个鉴定结果，裂殖壶HS01为裂殖壶(Schizoochytrium limacinum)。Based on the above respective identification results, the Schizochytrium HS01 is a Schizoochytrium limacinum.
三、裂殖壶HS01的保藏3. Deposit of the Schizontium HS01
裂殖壶(Schizoochytrium limacinum)HS01已于2017年03月10日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号)，保藏编号为CGMCC No.13746。Schizoochytrium limacinum HS01 was deposited on March 10, 2017 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) The number is CGMCC No.13746.
实施例2、裂壶藻粉的制备Example 2 Preparation of cracked pot algae powder
利用实施例1的裂壶藻HS01制备裂壶藻粉的步骤如下，实验重复三次，结果取平均值：The procedure for preparing the karst algae powder using the Schizochytrium sp. HS01 of Example 1 was as follows. The experiment was repeated three times, and the results were averaged:
一、培养基的制备First, the preparation of the medium
摇瓶培养基1由溶质和溶剂组成，溶剂为水，溶质及其浓度分别为60g/L葡萄糖、酵母膏5g/L；摇瓶培养基2由溶质和溶剂组成，溶剂为水，溶质及其浓度分别为150g/L葡萄糖、酵母膏25g/L。The shake flask medium 1 is composed of a solute and a solvent, the solvent is water, the solute and its concentration are respectively 60 g/L glucose, and the yeast extract is 5 g/L; the shake flask medium 2 is composed of a solute and a solvent, the solvent is water, the solute and The concentrations were 150 g/L glucose and yeast extract 25 g/L, respectively.
种子培养基1由溶质和溶剂组成，溶剂为水，溶质及其浓度分别为葡萄糖60g/L、酵母膏8g/L、酵母粉3g/L、Na 2SO 4 5g/L、KCl 0.5g/L、MgSO 4 1.0g/L、K 2SO 4 0.5g/L、KH 2PO 4 1.0g/L、(NH 4) 2SO 4 2.0g/L、CaCl 2 0.5g/L、CuSO 4 0.001g/L、ZnSO 4 0.001g/L，配置完成后利用碱(氢氧化钠溶液或是氨水)调节初始pH6.0；种子培养基2由溶质和溶剂组成，溶剂为水，溶质及其浓度分别为葡萄糖150g/L、酵母膏25g/L、酵母粉8g/L、Na 2SO 4 20g/L、KCl 1.5g/L、MgSO 4 3.0g/L、K 2SO 4 2.5g/L、KH 2PO 4 2.0g/L、(NH 4) 2SO 4 5.0g/L、CaCl 2 2.5g/L、CuSO 4 0.02g/L、ZnSO 4 0.02g/L，配置完成后利用碱(氢氧化钠溶液或是氨水)调节初始pH6.0。 Seed medium 1 consists of solute and solvent. The solvent is water. The solute and its concentration are glucose 60g/L, yeast extract 8g/L, yeast powder 3g/L, Na 2 SO 4 5g/L, KCl 0.5g/L. MgSO 4 1.0 g/L, K 2 SO 4 0.5 g/L, KH 2 PO 4 1.0 g/L, (NH 4 ) 2 SO 4 2.0 g/L, CaCl 2 0.5 g/L, CuSO 4 0.001 g/ L, ZnSO 4 0.001g / L, after the configuration is completed, the initial pH is adjusted by alkali (sodium hydroxide solution or ammonia water); the seed medium 2 is composed of solute and solvent, the solvent is water, the solute and its concentration are respectively glucose 150g/L, yeast extract 25g/L, yeast powder 8g/L, Na 2 SO 4 20g/L, KCl 1.5g/L, MgSO 4 3.0g/L, K 2 SO 4 2.5g/L, KH 2 PO 4 2.0g/L, (NH 4 ) 2 SO 4 5.0g/L, CaCl 2 2.5g/L, CuSO 4 0.02g/L, ZnSO 4 0.02g/L, after the completion of the configuration, use alkali (sodium hydroxide solution or Ammonia water) was adjusted to an initial pH of 6.0.
发酵培养基1由溶质和溶剂组成，溶剂为水，溶质及其浓度分别为葡萄糖60g/L、酵母膏8g/L、酵母粉3g/L、Na 2SO 4 5g/L、KCl 0.5g/L、MgSO 4 1.0g/L、K 2SO 4 0.5g/L、KH 2PO 4 1.0g/L、(NH 4) 2SO 4 2.0g/L、CaCl 2 0.5g/L、CuSO 4 0.001g/L、ZnSO 4 0.001g/L、生物素0.001g/L、玉米淀粉0.1g/L，配置完成后利用碱(氢氧化钠溶液或是氨水)调节初始pH6.0；发酵培养基2由溶质和溶剂组成，溶剂为水，溶质及其浓度分别为葡萄糖150g/L、酵母膏25g/L、酵母粉8g/L、Na 2SO 4 20g/L、KCl 1.5g/L、MgSO 4 3.0g/L、K 2SO 4 2.5g/L、KH 2PO 4 2.0g/L、(NH 4) 2SO 4 5.0g/L、CaCl 2 2.5g/L、CuSO 4 0.02g/L、ZnSO 4 0.02g/L、生物素0.06g/L、玉米 淀粉10g/L、豌豆蛋白粉20g/L，配置完成后利用碱(氢氧化钠溶液或是氨水)调节初始pH6.0；发酵培养基3由溶质和溶剂组成，溶剂为水，溶质及其浓度分别为葡萄糖60g/L、酵母膏8g/L、酵母粉3g/L、Na 2SO 4 5g/L、KCl 0.5g/L、MgSO 4 1.0g/L、K 2SO 4 0.5g/L、KH 2PO 4 1.0g/L、(NH 4) 2SO 4 2.0g/L、CaCl 2 0.5g/L、CuSO 4 0.001g/L、ZnSO 4 0.001g/L、生物素0.001g/L、辛烯基琥珀酸淀粉钠0.1g/L，配置完成后利用碱(氢氧化钠溶液或是氨水)调节初始pH6.0；发酵培养基4由溶质和溶剂组成，溶剂为水，溶质及其浓度分别为葡萄糖150g/L、酵母膏25g/L、酵母粉8g/L、Na 2SO 4 20g/L、KCl 1.5g/L、MgSO 4 3.0g/L、K 2SO 4 2.5g/L、KH 2PO 4 2.0g/L、(NH 4) 2SO 4 5.0g/L、CaCl 2 2.5g/L、CuSO 4 0.02g/L、ZnSO 4 0.02g/L、生物素0.06g/L、辛烯基琥珀酸淀粉钠10g/L、乳清蛋白粉20g/L，配置完成后利用碱(氢氧化钠溶液或是氨水)调节初始pH6.0。 Fermentation medium 1 consists of solute and solvent. The solvent is water. The solute and its concentration are glucose 60g/L, yeast extract 8g/L, yeast powder 3g/L, Na 2 SO 4 5g/L, KCl 0.5g/L. MgSO 4 1.0 g/L, K 2 SO 4 0.5 g/L, KH 2 PO 4 1.0 g/L, (NH 4 ) 2 SO 4 2.0 g/L, CaCl 2 0.5 g/L, CuSO 4 0.001 g/ L, ZnSO 4 0.001g / L, biotin 0.001g / L, corn starch 0.1g / L, after the completion of the configuration with alkali (sodium hydroxide solution or ammonia) to adjust the initial pH6.0; fermentation medium 2 by solute and Solvent composition, solvent is water, solute and its concentration are glucose 150g / L, yeast paste 25g / L, yeast powder 8g / L, Na 2 SO 4 20g / L, KCl 1.5g / L, MgSO 4 3.0g / L K 2 SO 4 2.5 g/L, KH 2 PO 4 2.0 g/L, (NH 4 ) 2 SO 4 5.0 g/L, CaCl 2 2.5 g/L, CuSO 4 0.02 g/L, ZnSO 4 0.02 g/ L, biotin 0.06g / L, corn starch 10g / L, pea protein powder 20g / L, after the configuration is completed with alkali (sodium hydroxide solution or ammonia) to adjust the initial pH6.0; fermentation medium 3 from the solute and solvent Composition, the solvent is water, the solute and its concentration are glucose 60g / L, yeast extract 8g / L, yeast powder 3g / L, Na 2 SO 4 5g / L, KCl 0.5 g/L, MgSO 4 1.0 g/L, K 2 SO 4 0.5 g/L, KH 2 PO 4 1.0 g/L, (NH 4 ) 2 SO 4 2.0 g/L, CaCl 2 0.5 g/L, CuSO 4 0.001g / L, ZnSO 4 0.001g / L, biotin 0.001g / L, sodium octenyl succinate 0.1g / L, after the completion of the configuration with alkali (sodium hydroxide solution or ammonia) to adjust the initial pH6.0 Fermentation medium 4 consists of solute and solvent. The solvent is water. The solute and its concentration are glucose 150g/L, yeast paste 25g/L, yeast powder 8g/L, Na 2 SO 4 20g/L, KCl 1.5g/ L, MgSO 4 3.0 g/L, K 2 SO 4 2.5 g/L, KH 2 PO 4 2.0 g/L, (NH 4 ) 2 SO 4 5.0 g/L, CaCl 2 2.5 g/L, CuSO 4 0.02 g /L, ZnSO 4 0.02g / L, biotin 0.06g / L, sodium octenyl succinate 10g / L, whey protein powder 20g / L, after the completion of the use of alkali (sodium hydroxide solution or ammonia) Adjust the initial pH to 6.0.
二、裂壶藻粉的制备及指标检测2. Preparation and index detection of cracked pot algae powder
1、制备1, preparation
将裂壶藻HS01接种至摇瓶培养基1中，在转速200rpm、温度20℃的条件下培养24h，得到摇瓶培养液1；将摇瓶培养液1接种至种子培养基1中，在溶氧为10～80％(生长过程溶解氧是一个动态过程)、温度20℃的条件下培养48h，在培养过程中保持培养液的pH为4.5～6.5之间，发酵过程pH会下降，通过氨水或是氢氧化钠溶液进行调节，得到种子培养液1；将种子培养液1按照10％的接种量接种至发酵培养基1中，在溶氧10～80％(生长过程溶解氧是一个动态的过程)、温度20℃的条件下培养120h，得到发酵液，将该发酵液记为发酵液1，在培养过程中保持培养液的pH为4.5～6.5之间，发酵过程pH会下降，通过氨水或是氢氧化钠溶液进行调节。The Physalis algae HS01 was inoculated into the shake flask medium 1, and cultured at a rotation speed of 200 rpm and a temperature of 20 ° C for 24 hours to obtain a shake flask culture solution 1; the shake flask culture solution 1 was inoculated into the seed culture medium 1 to be dissolved. Oxygen is 10 to 80% (dissolved oxygen is a dynamic process during growth), cultured at a temperature of 20 ° C for 48 h, and the pH of the culture solution is maintained between 4.5 and 6.5 during the cultivation, and the pH of the fermentation process is lowered. Or the sodium hydroxide solution is adjusted to obtain the seed culture solution 1; the seed culture solution 1 is inoculated into the fermentation medium 1 according to the 10% inoculation amount, and the dissolved oxygen is 10 to 80% (the dissolved oxygen in the growth process is a dynamic The process is carried out at a temperature of 20 ° C for 120 hours to obtain a fermentation broth. The fermentation broth is recorded as the fermentation broth 1 , and the pH of the culture solution is maintained between 4.5 and 6.5 during the cultivation, and the pH of the fermentation process is lowered. Or adjust with sodium hydroxide solution.
将裂壶藻HS01接种至摇瓶培养基2中，在转速400rpm、温度30℃的条件下培养48h，得到摇瓶培养液2；将摇瓶培养液2接种至种子培养基2中，在溶氧为10～80％、温度30℃的条件下培养24h，在培养过程中保持培养液的pH为4.5～6.5之间，发酵过程pH会下降，通过氨水或是氢氧化钠溶液进行调节，得到种子培养液2；将种子培养液2按照20％的接种量接种至发酵培养基2中，在溶氧10～80％、温度30℃的条件下培养72h，得到发酵液，将该发酵液记为发酵液2，在培养过程中保持培养液的pH为4.5～6.5之间，发酵过程pH会下降，通过氨水或是氢氧化钠溶液进行调节。The Physalis algae HS01 was inoculated into the shake flask medium 2, and cultured at a rotation speed of 400 rpm and a temperature of 30 ° C for 48 hours to obtain a shake flask culture solution 2; the shake flask culture solution 2 was inoculated into the seed culture medium 2, and dissolved therein. The culture is carried out for 24 hours under the condition of oxygen at 10 to 80% and temperature of 30 ° C. The pH of the culture solution is maintained between 4.5 and 6.5 during the cultivation, and the pH of the fermentation process is lowered, and the pH is adjusted by ammonia or sodium hydroxide solution. Seed culture solution 2; seed culture solution 2 was inoculated into fermentation medium 2 according to 20% inoculation amount, and cultured under the conditions of dissolved oxygen of 10 to 80% and temperature of 30 ° C for 72 hours to obtain a fermentation liquid, and the fermentation liquid was recorded. For the fermentation broth 2, the pH of the culture solution is maintained between 4.5 and 6.5 during the cultivation, and the pH of the fermentation process is lowered, and the pH is adjusted by ammonia water or sodium hydroxide solution.
将裂壶藻HS01接种至摇瓶培养基1中，在转速200rpm、温度20℃的条件下培养24h，得到摇瓶培养液1；将摇瓶培养液1接种至种子培养基1中，在溶氧为10～80％、温度20℃的条件下培养48h，在培养过程中保持培养液的pH为4.5～6.5之间，发酵过程pH会下降，通过氨水或是氢氧化钠溶液进行调节，得到种子培养液1；将种子培养液1按照10％的接种量接种至发酵培养基3中，在溶氧0～80％、温度20℃的条件下培养120h，得到发酵液，将该发酵液记为发酵液3，在培养过程中保持培养液的pH 4.5～6.5之间，发酵过程pH会下降，通过氨水或是氢氧化 钠溶液进行调节。The Physalis algae HS01 was inoculated into the shake flask medium 1, and cultured at a rotation speed of 200 rpm and a temperature of 20 ° C for 24 hours to obtain a shake flask culture solution 1; the shake flask culture solution 1 was inoculated into the seed culture medium 1 to be dissolved. Oxygen is 10～80%, and the temperature is 20°C for 48h. During the culture process, the pH of the culture solution is maintained between 4.5 and 6.5. The pH of the fermentation process will decrease and it will be adjusted by ammonia or sodium hydroxide solution. Seed culture solution 1; seed culture solution 1 was inoculated into fermentation medium 3 at a seeding rate of 10%, and cultured under the conditions of 0 to 80% dissolved oxygen at a temperature of 20 ° C for 120 hours to obtain a fermentation liquid, and the fermentation liquid was recorded. For the fermentation liquid 3, the pH of the culture solution is maintained between 4.5 and 6.5 during the cultivation, and the pH of the fermentation process is lowered, and the pH is adjusted by ammonia water or sodium hydroxide solution.
将裂壶藻HS01接种至摇瓶培养基2中，在转速400rpm、温度30℃的条件下培养24h，得到摇瓶培养液2；将摇瓶培养液2接种至种子培养基2中，在溶氧为10～80％、温度30℃的条件下培养24h，在培养过程中保持培养液的pH4.5～6.5之间，发酵过程pH会下降，通过氨水或是氢氧化钠溶液进行调节，得到种子培养液2；将种子培养液2按照20％的接种量接种至发酵培养基4中，在溶氧10～80％、温度30℃的条件下培养72h，得到发酵液，将该发酵液记为发酵液4，在培养过程中保持培养液的pH4.5～6.5之间，发酵过程pH会下降，通过氨水或是氢氧化钠溶液进行调节。The Physalis algae HS01 was inoculated into the shake flask medium 2, and cultured under the conditions of a rotation speed of 400 rpm and a temperature of 30 ° C for 24 hours to obtain a shake flask culture solution 2; the shake flask culture solution 2 was inoculated to the seed culture medium 2, and dissolved therein. Oxygen is 10 to 80%, and the temperature is 30 ° C for 24 h. During the cultivation, the pH of the culture solution is maintained between 4.5 and 6.5, the pH of the fermentation process is lowered, and the ammonia or water is adjusted to obtain Seed culture solution 2; seed culture solution 2 was inoculated into fermentation medium 4 according to the inoculation amount of 20%, and cultured under the conditions of dissolved oxygen of 10 to 80% and temperature of 30 ° C for 72 hours to obtain a fermentation liquid, and the fermentation liquid was recorded. For the fermentation broth 4, the pH of the culture solution is maintained between 4.5 and 6.5 during the cultivation, and the pH of the fermentation process is lowered, and the pH is adjusted by the ammonia water or the sodium hydroxide solution.
发酵结束后，向发酵液1中添加抗氧化剂1(抗氧化剂1由天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸组成，其中，天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸的质量比为20:2:10:10:2)得到混合液，该混合液中天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸的质量百分比含量分别为0.2％、0.02％、0.1％、0.1％和0.02％，将该混合液经过乳化混匀，获得稳定的发酵液1；将稳定的发酵液1进行巴氏灭菌，然后进行喷雾、滚筒或是冷冻干燥制备获得裂壶藻粉1。After the fermentation, the antioxidant 1 is added to the fermentation liquid 1 (the antioxidant 1 is composed of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid, among which natural mixed tocopherol, rosemary, tea The mass ratio of polyphenols, isoascorbic acid and phytic acid is 20:2:10:10:2) to obtain a mixed liquid in which the quality of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid The percentage content is 0.2%, 0.02%, 0.1%, 0.1%, and 0.02%, respectively, and the mixture is emulsified and mixed to obtain a stable fermentation liquid 1; the stable fermentation liquid 1 is pasteurized, and then sprayed. The cracked pot algae powder 1 was prepared by roller or freeze-drying.
向发酵液2中添加抗氧化剂2(抗氧化剂2由天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸组成，其中，天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸的质量比为40:3:20:20:4)得到混合液，该混合液中天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸的质量百分比含量分别为0.4％、0.03％、0.2％、0.2％和0.04％，将该混合液经过乳化混匀，获得稳定的发酵液2；将稳定的发酵液2进行巴氏灭菌，然后进行喷雾、滚筒或是冷冻干燥制备获得裂壶藻粉2。Adding antioxidant 2 to fermentation broth 2 (antioxidant 2 consists of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid, among which natural mixed tocopherol, rosemary, tea polyphenol, and different The mass ratio of ascorbic acid to phytic acid is 40:3:20:20:4) to obtain a mixed liquid, and the mass percentage of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid in the mixture is respectively 0.4%, 0.03%, 0.2%, 0.2%, and 0.04%, the mixture is emulsified and mixed to obtain a stable fermentation liquid 2; the stable fermentation liquid 2 is pasteurized, and then sprayed, drum or The cleavage algae powder 2 was obtained by freeze-drying.
将发酵液3经过离心收集裂壶藻细胞泥，按照裂壶藻细胞泥体积加入相同体积的无菌去离子水混匀，然后进行离心，重复清洗2～3次，获得裂壶藻细胞泥；向该裂壶藻细胞泥中添加抗氧化剂3(抗氧化剂3由天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸组成，其中，天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸的质量比为60:2:40:30:6)得到混合物，该混合物中天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸的质量百分比含量分别为0.6％、0.02％、0.4％、0.3％和0.06％，将该混合物经过乳化混匀，获得稳定的细胞泥；将该稳定的细胞泥经过巴氏灭菌，然后进行干燥(喷雾、滚筒或是冷冻干燥，三者选一)制备获得裂壶藻粉，将该裂壶藻粉记为裂壶藻粉3。The fermentation broth 3 is centrifuged to collect the cell mud of the phloem, and the same volume of sterile deionized water is added according to the volume of the granules of the phloem, and then centrifuged, and the washing is repeated 2 to 3 times to obtain the cell mud of the karyophylla; Adding antioxidant 3 to the cell salt of the karst algae (antioxidant 3 consists of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid, among which natural mixed tocopherol, rosemary, tea The mass ratio of phenol, isoascorbic acid and phytic acid is 60:2:40:30:6), and the mass percentage of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid in the mixture are respectively 0.6%, 0.02%, 0.4%, 0.3%, and 0.06%, the mixture is emulsified and mixed to obtain a stable cell sludge; the stabilized cell sludge is pasteurized and then dried (spray, roller or It is freeze-dried, and the three are selected to obtain the cracked pot algae powder, and the cracked pot algae powder is recorded as the cracked pot algae powder 3.
将发酵液4经过离心收集裂壶藻细胞泥，按照裂壶藻细胞泥体积加入相同体积的无菌去离子水混匀，然后进行离心，重复清洗2～3次，获得裂壶藻细胞泥；向该裂壶藻细胞泥中添加抗氧化剂4(抗氧化剂4由天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸组成，其中，天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸的质量比为80:2:40:40:8)得到混合物，该混合物中天然混合生育酚、迷迭香、茶多酚、异抗坏血酸和植酸的质量百分比含量分别为0.8％、0.02％、0.4％、 0.4％和0.08％，将该混合物经过乳化混匀，获得稳定的细胞泥；将该稳定的细胞泥经过巴氏灭菌，然后进行干燥(喷雾、滚筒或是冷冻干燥，三者选一)制备获得裂壶藻粉，将该裂壶藻粉记为裂壶藻粉4。The fermentation broth 4 is centrifuged to collect the cell mud of the karyophylla, and the same volume of sterile deionized water is added according to the volume of the granules of the phloem, and then centrifuged, and the washing is repeated 2 to 3 times to obtain the cell mud of the karyophylla; Adding antioxidant 4 to the cell salt of the karst algae (antioxidant 4 consists of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid, among which natural mixed tocopherol, rosemary, tea The mass ratio of phenol, isoascorbic acid and phytic acid is 80:2:40:40:8), and the mass percentage of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid in the mixture are respectively 0.8%, 0.02%, 0.4%, 0.4%, and 0.08%, the mixture is emulsified and mixed to obtain a stable cell sludge; the stabilized cell sludge is pasteurized and then dried (spray, roller or It is freeze-dried, and the three are selected to obtain the cracked pot algae powder, and the cracked pot algae powder is recorded as the cracked pot algae powder 4.
2、指标检测2, indicator detection
分别检测步骤1得到的裂壶藻粉1-4的蛋白质、水分、脂肪酸、灰分、DHA的含量，具体检测方法如下：The protein, moisture, fatty acid, ash and DHA content of the Phytophthora powder 1-4 obtained in the step 1 were respectively detected, and the specific detection methods are as follows:
蛋白质的检测：按照GB 5009.9《食品安全国家标准食品中蛋白质的测定》进行。Detection of protein: According to GB 5009.9 "Food Safety National Standard Food Determination of Protein".
水分的检测：按照GB 5009.3《食品安全国家标准食品中水分的测定》进行。Moisture detection: According to GB 5009.3 "Food Safety National Standard for Determination of Moisture in Food".
灰分的检测：按照GB 5009.4《食品安全国家标准食品中灰分的测定》进行。Detection of ash: According to GB 5009.4 "Determination of ash in foods in national food safety standards".
脂肪酸的检测：按照GB 5009.168《食品安全国家标准食品中脂肪酸的测定》进行。Detection of fatty acids: in accordance with GB 5009.168 "Food Safety National Standard Food Determination of Fatty Acids in Foods".
DHA的检测：按照GB 26400《食品安全国家标准食品添加剂二十二碳六烯酸油脂(发酵法)》进行。Detection of DHA: According to GB 26400 "Food Safety National Standard Food Additive Twenty Hexahexaenoic Acid (Fermentation Method)".
牛奶中Sn-2位DHA占总DHA的比例检测：脂肪酸组成按照GB 5009.168-2016《食品安全国家标准食品中脂肪酸的测定》和GB/T 24894-2010/ISO 6800:1997方法进行。Determination of the ratio of DHA to total DHA in milk: The fatty acid composition was carried out according to GB 5009.168-2016 "Food Safety National Standard Food Determination of Fatty Acids" and GB/T 24894-2010/ISO 6800:1997 method.
结果显示，上述步骤中得到的裂壶藻粉中蛋白质的质量含量为10～60％，水分的质量含量为0.5～3.0％，灰分的质量含量为3～12％，脂肪酸的质量含量为25～50％。在脂肪酸中，不饱和脂肪酸DHA的质量含量为10～24％，DPA的质量含量为2.0～6.0％，EPA的质量含量为0.1～0.5％。The results show that the protein content of the cracked pot algae powder obtained in the above step is 10 to 60%, the water content is 0.5 to 3.0%, the mass content of the ash is 3 to 12%, and the mass content of the fatty acid is 25 to 50%. In the fatty acid, the unsaturated fatty acid DHA has a mass content of 10 to 24%, the DPA has a mass content of 2.0 to 6.0%, and the EPA has a mass content of 0.1 to 0.5%.
表1、裂壶藻粉的指标检测结果Table 1. Index test results of the cracked pot algae powder
Figure PCTCN2018115327-appb-000001
Figure PCTCN2018115327-appb-000001
注：表1中各指标的含量是指各物质占干粉的质量百分比；其他脂肪酸是指除C8:0辛酸、C10:0葵酸、C16:0棕榈酸、C18:0硬脂酸、C22:5 DPA、C20:5 EPA和C22:6 DHA外的脂肪酸。Note: The content of each indicator in Table 1 refers to the mass percentage of each substance in dry powder; other fatty acids refer to C8:0 octanoic acid, C10:0 acid, C16:0 palmitic acid, C18:0 stearic acid, C22: 5 DPA, C20: 5 EPA and C22: 6 fatty acids outside DHA.
三、裂壶藻粉的瘘管实验测试Third, the experimental test of the tube of algae
采用尼龙袋法进行裂壶藻粉1和2的过奶牛瘤胃瘘管实验。其操作步骤如下：The rumen fistula experiment of the cows with the cracked pot algae powders 1 and 2 was carried out by the nylon bag method. The steps are as follows:
1、试验动物与日粮1. Test animals and diet
装有永久性胃瘘管的奶牛(荷斯坦奶牛)。预饲期7天，在此期间进行驱虫，以1％的敌百虫药液驱除体外寄生虫，口服0.8mg/kg体重的盐酸左旋咪唑驱除体内寄生虫。在营养水平为1.3倍维持需要的条件下饲养，每天两次等量饲喂，07：00和16：00各喂一次，饲后自由饮水。A cow (Holstein cow) with a permanent gastric fistula. During the pre-feeding period of 7 days, deworming was carried out during this period, and the ectoparasite was removed by 1% trichlorfon solution, and the parasite was removed by oral administration of 0.8 mg/kg of levamisole hydrochloride. Raise at a nutritional level of 1.3 times to maintain the required conditions, feed twice a day, feed once at 07:00 and 16:00, and drink freely after feeding.
2、试样的制备2. Preparation of sample
裂壶藻粉采用“四分法”随机采集样本，65℃烘至恒重后，装入磨口瓶备用。The cracked pot algae powder was randomly collected by the "quadruple method", and dried at 65 ° C to a constant weight, and then placed in a grinding bottle for use.
3、尼龙袋的制作3, the production of nylon bags
用300目尼龙布，裁成170mm×130mm的长方形，对折后用涤纶线缝双道，制成大小为120mm×80mm的尼龙袋，散边用烙铁烫平。试验前将尼龙袋放入瘤胃平衡72h取出洗净烘干，检查无破损方可使用。A 300-mesh nylon cloth was cut into a rectangular shape of 170 mm × 130 mm, and after folding, a nylon bag having a size of 120 mm × 80 mm was formed by double-stitching with a polyester thread, and the loose side was flattened with a soldering iron. Before the test, put the nylon bag into the rumen balance for 72 hours, take it out and wash it, and check it without damage before use.
4、试验设计与测定方法4. Test design and measurement method
尼龙袋法瘤胃降解按奶牛饲养标准科研协作组等提出的方案进行，试验采用随机单位组设计，每只牛每一时间点设两个重复。The rumen degradation of the nylon bag method was carried out according to the scheme proposed by the Dairy Cow Breeding Standard Scientific Research Cooperation Group. The test was designed using a random unit group, and each cow was given two repetitions at each time point.
每个尼龙袋装入裂壶藻粉10g左右，试样方差分析差异不显著(P＞0.05)。每2袋牢系在一根30cm长的半聚乙烯管上，于早晨饲后2h将尼龙袋放置在瘤胃腹囊部，管的另一端挂在瘘管盖上。每只牛瘤胃内同时放6根管，共12袋。分别于放袋后0h、2h、4h、6h、12h、24h、36h及48h 8个时间点从每头牛的瘤胃中各取出一根管，用清水洗净，放入洗衣机中漂洗7分钟，至水澄清，然后在65℃下烘至恒重，并称重。Each nylon bag was filled with 10g of cracked pot algae powder, and the variance analysis of the sample was not significant (P>0.05). Each 2 bags were tied to a 30 cm long semi-polyethylene tube. The nylon bag was placed in the ventral sac of the rumen 2 hours after the morning feeding, and the other end of the tube was hung on the fistula cap. Six tubes were placed in the rumen of each cow for a total of 12 bags. Take a tube from each rumen of each cow at 0h, 2h, 4h, 6h, 12h, 24h, 36h and 48h after bag release, wash with water, and rinse in the washing machine for 7 minutes. Clarify to water, then bake at 65 ° C to constant weight and weigh.
干物质(DM)的测定按GB6435－86方法进行，DHA的测定由厦门汇盛生物有限公司负责。样品某时间点(t)的某养分降解率/％＝(1－残留的某养分质量/放入袋内的某养分总质量)×100％。The determination of dry matter (DM) was carried out according to the method of GB6435-86, and the determination of DHA was carried out by Xiamen Huisheng Biological Co., Ltd. The rate of degradation of a nutrient at a certain point (t) of the sample /% = (1 - the quality of a certain nutrient / the total mass of a nut in the bag) × 100%.
结果如表2所示，结果显示裂壶藻粉过奶牛瘤胃的最高降解率为54.7％。The results are shown in Table 2. The results showed that the highest degradation rate of the rumen of the clams was over 54.7%.
表2、裂壶藻粉的瘘管实验结果Table 2. Results of the fistula test of the cracked pot algae powder
Figure PCTCN2018115327-appb-000002
Figure PCTCN2018115327-appb-000002
Figure PCTCN2018115327-appb-000003
Figure PCTCN2018115327-appb-000003
实施例3、实施例2的裂壶藻粉可以提高牛奶中DHA含量The cracking pot algae powder of Example 3 and Example 2 can increase the DHA content in milk.
本实施例通过对奶牛饲喂实施例2的裂壶藻粉，检测牛奶中的DHA含量，确定裂壶藻粉对牛奶中DHA含量的影响，实验重复三次。In this example, the cows were fed the cracked pot algae powder of Example 2, and the DHA content in the milk was measured to determine the effect of the cracked pot algae powder on the DHA content in the milk. The experiment was repeated three times.
一、饲养方法：First, the feeding method:
选取体重、月龄均无显著差异的健康荷斯坦奶牛60头，随机分为六组，每组10头，即散养实验组1、散养实验组2、散养空白对照组、圈养实验组1、圈养实验组2和圈养空白对照组。Sixty healthy Holstein cows with no significant differences in body weight and age were randomly divided into six groups, 10 in each group, namely, free-range experimental group 1, free-range experimental group 2, free-range blank control group, and captive experimental group. 1. Captive experimental group 2 and captive blank control group.
首先对各实验组奶牛进行预饲期养殖(预饲期为15天，根据奶牛的适应情况逐步加大量裂壶藻粉喂养量)；之后的正式喂养期将裂壶藻粉按照一定的添加比例投入饲料中进行搅拌混合，每隔8小时饲喂一次。Firstly, the pre-feeding period of the cows in each experimental group was carried out (the pre-feeding period was 15 days, and the amount of cracked pot algae powder was gradually added according to the adaptation of the cows); after the formal feeding period, the cracked pot algae powder was added according to a certain proportion. Stir in the feed and mix and feed once every 8 hours.
散养实验组1和散养实验组2的饲料中添加的裂壶藻粉分别为实施例1的裂壶藻粉1和2，圈养实验组1和圈养实验组2的饲料中添加的裂壶藻粉分别为实施例1的裂壶藻粉1和2，具体饲喂方法如下：The cracked pot algae powder added to the feed of the free-range experiment group 1 and the free-range experiment group 2 was the crack pot of the cracked pot algae powders 1 and 2 of Example 1, the captive experiment group 1 and the captive experiment group 2, respectively. The algal flours were respectively the cracked pot algae powders 1 and 2 of Example 1, and the specific feeding methods were as follows:
预饲期：实验组饲喂裂壶藻粉的量逐渐增加，具体如下：Pre-feeding period: The amount of algae powder fed to the experimental group is gradually increased, as follows:
预饲期第1和2天裂壶藻粉的饲喂量为50mg/天/头；The feeding amount of the cracked pot algae powder on the first and second days of the pre-feeding period was 50 mg/day/head;
预饲期第3和4天裂壶藻粉的饲喂量为75mg/天/头；The feeding amount of the cracked pot algae powder on the 3rd and 4th days of the pre-feeding period was 75 mg/day/head;
预饲期第5和6天裂壶藻粉的饲喂量为100mg/天/头；The feeding amount of the cracked pot algae powder on the 5th and 6th day of the pre-feeding period was 100 mg/day/head;
预饲期第7和8天裂壶藻粉的饲喂量为125mg/天/头；The feeding amount of the cracked pot algae powder on the 7th and 8th days of the pre-feeding period was 125 mg/day/head;
预饲期第9和10天裂壶藻粉的饲喂量为150mg/天/头；The feeding amount of the cracked pot algae powder on the 9th and 10th days of the pre-feeding period was 150 mg/day/head;
预饲期第11和12天裂壶藻粉的饲喂量为200mg/天/头；The feeding amount of the cracked pot algae powder on the 11th and 12th days of the pre-feeding period was 200 mg/day/head;
预饲期第13、14和15天裂壶藻粉的饲喂量为250mg/天/头。The feeding amount of the cracked pot algae powder on the 13th, 14th and 15th days of the pre-feeding period was 250 mg/day/head.
正式喂养期为裂壶藻粉250mg/天/头。The formal feeding period is 250 mg/day/head of the cracked pot algae powder.
空白对照组奶牛饲喂的饲料中不添加裂壶藻粉，其余成分与实验组相同，饲喂时间和饲喂量均同实验组。In the blank control group, the feed of the cows was not added with the cracked pot algae powder, and the other components were the same as the experimental group. The feeding time and feeding amount were the same as the experimental group.
散养实验组1、散养实验组2和散养空白对照组的奶牛进行散养，散养场地中无奶牛其他可食食物；圈养实验组1、圈养实验组2和圈养空白对照组的奶牛进行圈养，圈中无其他可食食物。The free-range experimental group 1, the free-range experimental group 2 and the free-range blank control group of the dairy cows were randomly reared, and there were no other edible foods for the dairy cows in the free-range field; the captive experimental group 1, the captive experimental group 2 and the captive blank control group of cows In captivity, there are no other edible foods in the circle.
二、牛奶数据检测Second, milk data detection
DHA奶样采集：采集早中晚三次所有饲喂牛的奶样，混合抽样送检；并根据国标GB5413.27-2010婴幼儿食品和乳品中脂肪酸的测定；检测牛奶中的乳脂、乳蛋白、DHA含量指标、牛奶中Sn-2位DHA占总DHA的比例。DHA milk sample collection: collect all the milk samples of cattle fed three times in the morning, evening, and mixed samples for inspection; and according to the national standard GB5413.27-2010 determination of fatty acids in infant food and dairy products; detect milk fat, milk protein, The DHA content index and the ratio of DH in the Sn-2 position in the milk to the total DHA.
结果(表3)显示，无论是散养还是圈养，实验组牛奶中的乳脂和乳蛋白的含 量均同空白对照组无显著差异。而饲料中添加实施例1的裂壶藻粉后，随着饲喂时间的增加，牛奶中的DHA含量逐渐增加，且各实验组牛奶中DHA含量均显著高于对照组，两个对照组牛奶中DHA含量无显著差异。The results (Table 3) showed that the milk fat and milk protein content in the experimental group were not significantly different from the blank control group, either in free or in captivity. When the algae powder of Example 1 was added to the feed, the DHA content in the milk gradually increased with the increase of feeding time, and the DHA content in the milk of each experimental group was significantly higher than that of the control group. There was no significant difference in the content of DHA.
表3、牛奶DHA含量检测结果(mg/100g牛奶)Table 3, milk DHA content test results (mg / 100g milk)
Figure PCTCN2018115327-appb-000004
Figure PCTCN2018115327-appb-000004
注：预饲期结束是预饲期第15天，正式期第7、15、30、45、60和75天分别为正式喂养期的第7、15、30、45、60和75天，正式期75天后是指第100天。Note: The end of the pre-feeding period is the 15th day of the pre-feeding period, and the 7th, 15th, 30th, 45th, 60th and 75th days of the official period are the 7th, 15th, 30th, 45th, 60th and 75th days of the official feeding period, respectively. After 75 days, it refers to the 100th day.
表4、牛奶中Sn-2位DHA占总DHA比例(％)Table 4, the ratio of DHA to total DHA in the Sn-2 position in milk (%)
Figure PCTCN2018115327-appb-000005
Figure PCTCN2018115327-appb-000005
注：散养和圈养在“牛奶中Sn-2位DHA占总DHA比例”方面没有明显差别，而且其比例一般都在30～95％范围波动并没有特殊的规律。Note: There is no significant difference between the free-range and captive in the “DHA ratio of total DHA in the Sn-2 position in milk”, and the proportion is generally fluctuated in the range of 30 to 95% with no special rules.
实施例4、实施例2的裂壶藻粉可以提高鸡蛋品质及蛋鸡的产蛋性能The cracked pot algae powder of Example 4 and Example 2 can improve egg quality and laying performance of laying hens
本实施例通过对蛋鸡饲喂实施例2的裂壶藻粉，检测裂壶藻粉对蛋鸡产蛋性能及鸡蛋品质的影响。In this example, the laying hen powder of Example 2 was fed to the laying hen to detect the effect of the cracked pot algae powder on laying performance and egg quality of the laying hen.
1、试验动物1. Test animals
随机选取健康体重无显著差异的处于产蛋期的蛋鸡(海兰白鸡)360只，各蛋鸡间的月龄无显著差异，将蛋鸡随机分为4组(对照组，0.5％实验组，1.0％实 验组和1.5％实验组)，每组6个平行，每个平行15只。A total of 360 laying hens (Hailan white chicken) with no significant difference in healthy body weight were randomly selected. There was no significant difference in age between the laying hens. The laying hens were randomly divided into 4 groups (control group, 0.5% experiment). Group, 1.0% experimental group and 1.5% experimental group), each group of 6 parallel, each parallel 15 .
2、饲养管理2, feeding management
采用3层笼养，持续光照。试验日粮以干粉料形式饲喂，日喂3次，自由采食和饮水，按笼记录每日采食量。实验组蛋鸡基础日粮为玉米-豆粕型日粮，各实验组饲喂添加了实施例2的裂壶藻粉的基础日粮，0.5％实验组的食物中裂壶藻粉1的质量含量为0.5％，1.0％实验组的食物中裂壶藻粉1的质量含量为1.0％，1.5％实验组的食物中裂壶藻粉1的质量含量为1.5％；对照组饲喂基础日粮。将实验组饲喂含有裂壶藻粉的当天记为第1天。每天统计产蛋率，分别在第15天和第25天检测鸡蛋的胆固醇含量(第25天的结果如表5所示)，并计算产蛋率、鸡蛋胆固醇和DHA含量的变化，结果如表6所示。It is housed in 3 layers and is continuously illuminated. The test diet was fed as a dry powder, fed three times a day, fed ad libitum and drinking water, and the daily feed intake was recorded by cage. In the experimental group, the basic diet of the laying hens was a corn-soybean meal type diet. Each experimental group was fed with the basic diet supplemented with the cracked pot algae powder of Example 2, and the mass content of the cracked pot algae powder 1 in the 0.5% experimental group. For the 0.5%, 1.0% of the experimental group, the mass content of the cracked pot algae powder was 1.0%, and 1.5% of the food in the experimental group was 1.5%; the control group was fed the basal diet. The day when the experimental group was fed with the cracked pot algae powder was recorded as the first day. The egg production rate was measured daily, and the cholesterol content of the eggs was measured on the 15th day and the 25th day respectively (the results on the 25th day are shown in Table 5), and the changes in egg production rate, egg cholesterol and DHA content were calculated. 6 is shown.
表5、裂壶藻粉1饲养第25天产蛋率、胆固醇和DHA含量的测定结果Table 5: Determination of egg production rate, cholesterol and DHA content on the 25th day of cultivating
Figure PCTCN2018115327-appb-000006
Figure PCTCN2018115327-appb-000006
Figure PCTCN2018115327-appb-000007
Figure PCTCN2018115327-appb-000007
表6、饲养第15天和第25天产蛋率及鸡蛋营养成分的变化Table 6. Changes in egg production rate and egg nutrient composition on the 15th and 25th day of feeding
Figure PCTCN2018115327-appb-000008
Figure PCTCN2018115327-appb-000008
注：表6中，“产蛋率增减”是指与对照组同时期蛋鸡的产蛋率相比的变化量，“胆固醇增减”是指与对照组同时期鸡蛋的胆固醇含量相比的变化量，“+”表示增加，“-”表示减少。Note: In Table 6, “increased egg production rate” refers to the change in egg production rate of the laying hens in the same period of the control group. “Cholesterol increase and decrease” refers to the cholesterol content of the eggs in the same period as the control group. The amount of change, "+" means increase, and "-" means decrease.
统计第25天的产蛋率、蛋重、采食量、蛋料比，检测鸡蛋脂质含量以及胆固醇含量，结果如表7所示。The egg production rate, egg weight, feed intake, and egg-to-feed ratio on the 25th day were measured, and the egg lipid content and cholesterol content were measured. The results are shown in Table 7.
表7、饲养不同量裂壶藻粉第25天对蛋鸡生产性能和脂质的影响Table 7. Effect of feeding the different amount of cracked pot algae powder on the production performance and lipid of laying hens
Figure PCTCN2018115327-appb-000009
Figure PCTCN2018115327-appb-000009
对各组间各项目(指标)进行显著性分析，其中产蛋率的显著性分析如下：Significant analysis was carried out for each item (indicator) between the groups, and the significant analysis of the egg production rate was as follows:
0.5％组统计量0.5% group statistic
组别Group NN 均值Mean 标准差Standard deviation 均值的标准差Standard deviation of mean
对照组Control group 66 91.60091.600 3.47743.4774 1.41961.4196
0.5％组0.5% group 66 87.61787.617 4.44414.4441 1.81431.8143
0.5％组独立样本检验0.5% independent sample test
Figure PCTCN2018115327-appb-000010
Figure PCTCN2018115327-appb-000010
1.0％组统计量1.0% group statistic
组别Group NN 均值Mean 标准差Standard deviation 均值的标准差Standard deviation of mean
对照Control 66 91.60091.600 3.47743.4774 1.41961.4196
1.0％组1.0% group 66 95.60095.600 1.85801.8580 .7585.7585
1.0％组独立样本检验1.0% independent sample test
Figure PCTCN2018115327-appb-000011
Figure PCTCN2018115327-appb-000011
1.5％组统计量1.5% group statistic
组别Group NN 均值Mean 标准差Standard deviation 均值的标准差Standard deviation of mean
对照Control 66 91.60091.600 3.47743.4774 1.41961.4196
1.5％组1.5% group 66 93.50093.500 2.13922.1392 .8733.8733
1.5％组独立样本检验1.5% independent sample test
Figure PCTCN2018115327-appb-000012
Figure PCTCN2018115327-appb-000012
从显著性分析可以看出，对于产蛋率，第25天0.5％组和1.5％组其产蛋率相对于对照组差异不显著(P＞0.05)，但是1.0％组能够显著提高蛋鸡的产蛋率(P＜0.05)，表明饲喂特定量的裂壶藻粉1可以提高蛋鸡的产蛋率。From the significance analysis, it can be seen that for the egg production rate, the egg production rate in the 0.5% group and the 1.5% group on the 25th day was not significantly different from the control group (P>0.05), but the 1.0% group could significantly improve the laying hens. The egg production rate (P<0.05) indicates that feeding a specific amount of schistosomiasis powder 1 can increase the laying rate of laying hens.
蛋鸡饲喂裂壶藻粉1，饲喂第15天DHA含量相对对照组，0.5％组可以提高317.71％，达到150mg/100g；1.0％组可以提高579.48％，达到244mg/100g；而1.5％组可以提高885.79％，达到354mg/100g。饲喂25天后，0.5％组和1.0％组基本维持不变，而1.5％组有稍许下降，但与对照组相比，仍显著增加。表明，饲喂裂壶藻粉1可以提高蛋鸡鸡蛋中DHA含量，且食物中裂壶藻粉1添加量越多，鸡蛋中DHA含量越高。The laying hens were fed with the cracked pot algae powder 1. The DHA content on the 15th day of feeding was higher than that of the control group, and the 0.5% group could increase by 317.11% to 150mg/100g; the 1.0% group could increase by 579.48% to 244mg/100g; and 1.5%. The group can increase by 885.79% to 354 mg/100g. After 25 days of feeding, the 0.5% and 1.0% groups remained essentially unchanged, while the 1.5% group showed a slight decrease, but there was still a significant increase compared to the control group. It is indicated that feeding the cracked pot algae powder 1 can increase the DHA content in the egg of the laying hen, and the more the amount of the cracked pot algae powder 1 in the food, the higher the DHA content in the egg.
蛋鸡饲喂裂壶藻粉1，鸡蛋中胆固醇含量均明显下降；鸡蛋胆固醇的含量的下降量与食物中裂壶藻粉1的含量无明显关系，但随饲喂是时间的增加，鸡蛋中胆固醇含量有进一步下降的趋势。表明，饲喂裂壶藻粉1可以降低蛋鸡鸡蛋中胆固醇的含量。The laying hens fed the cracked pot algae powder 1 , the cholesterol content in the eggs decreased significantly; the amount of cholesterol in the egg had no significant relationship with the content of the cracked pot algae powder 1 in the food, but with the increase of time in the egg, in the egg There is a tendency for cholesterol levels to decline further. It is indicated that feeding the cracked pot algae powder 1 can reduce the cholesterol content in the egg of the laying hen.
按照上述方法，将上述步骤中的裂壶藻粉1替换为裂壶藻粉2，其他步骤均不变，得到了相同变化趋势的结果，表明，实施例1的裂壶藻粉1和2均具有相同的功能。According to the above method, the cracked pot algae powder 1 in the above step was replaced with the cracked pot algae powder 2, and the other steps were unchanged, and the same trend of change was obtained, indicating that the cracked pot algae powders 1 and 2 of Example 1 were both obtained. Has the same function.
工业应用Industrial application
本发明的裂壶藻(Schizochytrium limacinum)制备的裂壶藻粉，可以提高动物产品中DHA的含量，可以降低动物产品中的胆固醇含量，另外还可以提高家禽的产蛋性能。这种天然来源的高DHA含量动物产品，有机、安全、稳定、易吸收，可以作为人们摄取天然DHA的更安全有效途径，更能迎合和满足消费者需求，进而使本发明的裂壶藻及裂壶藻粉在普通食品、畜牧养殖领域具有广泛的应用。The Schizochytrium algae powder prepared by Schizochytrium limacinum of the present invention can increase the content of DHA in animal products, can lower the cholesterol content in animal products, and can also improve the egg laying performance of poultry. This natural source of high DHA content animal products, organic, safe, stable, easy to absorb, can be a safer and more effective way for people to ingest natural DHA, more able to cater to and meet consumer demand, and thus the Schizophrenia and the present invention The cracked pot algae powder has a wide range of applications in the fields of general food and animal husbandry.

Claims (28)

  1. 裂壶藻(Schizochytrium limacinum)或其制剂的下述任一应用：Any of the following applications of Schizochytrium limacinum or its preparation:
    A1)在提高动物产品品质中的应用；A1) Application in improving the quality of animal products;
    A2)在制备提高动物产品品质的物质中的应用；A2) application in the preparation of a substance for improving the quality of an animal product;
    A3)在提高动物产品产量中的应用；A3) Application in increasing the yield of animal products;
    A4)在制备提高动物产品产量的物质中的应用。A4) Use in the preparation of a substance which increases the yield of animal products.
  2. 根据权利要求1所述的应用，其特征在于：所述裂壶藻(Schizochytrium limacinum)为裂壶藻(Schizochytrium limacinum)HS01，所述裂壶藻(Schizochytrium limacinum)HS01在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.13746。The use according to claim 1, characterized in that the Schizochytrium limacinum is Schizochytrium limacinum HS01, and the Schizochytrium limacinum HS01 is in the Chinese Microbial Culture Collection Management Committee. The deposit number of the General Microbiology Center is CGMCC No. 13746.
  3. 根据权利要求1或2所述的应用，其特征在于：所述提高动物产品品质为提高所述动物产品DHA含量和/或提高所述动物产品Sn-2位DHA含量和/或降低所述动物产品胆固醇含量。The use according to claim 1 or 2, characterized in that said improving the quality of the animal product is to increase the DHA content of said animal product and/or to increase the DHA content of said animal product at the Sn-2 position and/or to lower said animal Product cholesterol content.
  4. 根据权利要求1-3中任一所述的应用，其特征在于：所述制剂为裂壶藻粉。The use according to any one of claims 1 to 3, characterized in that the preparation is a cracked algae powder.
  5. 根据权利要求1-4中任一所述的应用，其特征在于：所述制剂按照包括如下步骤的裂壶藻制剂的制备方法制备：培养权利要求1或2中所述的裂壶藻(Schizochytrium limacinum)，得到发酵液；利用所述发酵液制备得到所述制剂。The use according to any one of claims 1 to 4, characterized in that the preparation is prepared according to the preparation method of the Schizochytrium preparation comprising the steps of cultivating the Schizochytrium according to claim 1 or 2. Limacinum), a fermentation broth is obtained; the preparation is prepared using the fermentation broth.
  6. 根据权利要求5所述的应用，其特征在于：培养权利要求1或2中所述的裂壶藻(Schizochytrium limacinum)利用发酵培养基进行，所述发酵培养基由溶剂和溶质组成，所述溶剂为水，所述溶质及其浓度分别为葡萄糖60～150g/L、酵母膏8～25g/L、酵母粉3～8g/L、Na 2SO 4 5～20g/L、KCl 0.5～1.5g/L、MgSO 4 1.0～3.0g/L、K 2SO 4 0.5～2.5g/L、KH 2PO 4 1.0～2.0g/L、(NH 4) 2SO 4 2.0～5.0g/L、CaCl 2 0.5～2.5g/L、CuSO 4 0.001～0.02g/L、ZnSO 4 0.001～0.02g/L、生物素0.001～0.06g/L、淀粉0.1～10g/L和蛋白粉0～20g/L，pH为4.5～6.5。 The use according to claim 5, characterized in that the cultivation of the Schizochytrium limacinum according to claim 1 or 2 is carried out using a fermentation medium consisting of a solvent and a solute, the solvent For water, the solute and its concentration are glucose 60-150 g/L, yeast extract 8-25 g/L, yeast powder 3-8 g/L, Na 2 SO 4 5-20 g/L, KCl 0.5-1.5 g/ L, MgSO 4 1.0 to 3.0 g/L, K 2 SO 4 0.5 to 2.5 g/L, KH 2 PO 4 1.0 to 2.0 g/L, (NH 4 ) 2 SO 4 2.0 to 5.0 g/L, CaCl 2 0.5 ～2.5g/L, CuSO 4 0.001～0.02g/L, ZnSO 4 0.001～0.02g/L, biotin 0.001～0.06g/L, starch 0.1～10g/L and protein powder 0～20g/L, pH is 4.5 to 6.5.
  7. 根据权利要求5或6所述的应用，其特征在于：所述利用所述发酵液制备得到所述制剂包括：干燥所述发酵液，得到所述制剂。The use according to claim 5 or 6, wherein the preparation of the preparation using the fermentation broth comprises drying the fermentation broth to obtain the preparation.
  8. 根据权利要求5-7中任一所述的应用，其特征在于：所述方法包括在得到所述发酵液后向所述发酵液中添加抗氧化剂后再进行干燥得到所述制剂。The use according to any one of claims 5 to 7, characterized in that the method comprises adding an antioxidant to the fermentation broth after obtaining the fermentation broth, followed by drying to obtain the preparation.
  9. 根据权利要求8所述的应用，其特征在于：所述抗氧化剂为油溶抗氧化剂和/或水溶抗氧化剂。The use according to claim 8 wherein the antioxidant is an oil soluble antioxidant and/or a water soluble antioxidant.
  10. 根据权利要求9所述的应用，其特征在于：所述油溶抗氧化剂为迷迭香、天然混合生育酚、茶多酚和/或抗坏血酸棕榈酸酯；The use according to claim 9, wherein the oil-soluble antioxidant is rosemary, natural mixed tocopherol, tea polyphenol and/or ascorbyl palmitate;
    和/或，所述水溶抗氧化剂为植酸、抗坏血酸和/或异抗坏血酸。And/or the water soluble antioxidant is phytic acid, ascorbic acid and/or isoascorbic acid.
  11. 根据权利要求1-10中任一所述的应用，其特征在于：所述动物为a1) 或a2)或a3)：The use according to any one of claims 1 to 10, characterized in that the animal is a1) or a2) or a3):
    a1)家禽；A1) poultry;
    a2)鸡；A2) chicken;
    a3)北京白鸡、海兰白鸡、海兰褐蛋鸡或海兰粉壳鸡。A3) Beijing White Chicken, Hailan White Chicken, Hailan Brown Layer Chicken or Hailan Powder Shell Chicken.
  12. 根据权利要求11所述的应用，其特征在于：所述动物产品为所述动物产的蛋。The use according to claim 11 wherein said animal product is an egg produced by said animal.
  13. 根据权利要求1-10中任一所述的应用，其特征在于：所述动物为b1)或b2)：The use according to any one of claims 1 to 10, characterized in that the animal is b1) or b2):
    b1)反刍动物；B1) ruminants;
    b1)牛。B1) cattle.
  14. 根据权利要求13所述的应用，其特征在于：所述动物产品为所述动物的乳汁。The use according to claim 13 wherein said animal product is the milk of said animal.
  15. 权利要求5-10中任一所述裂壶藻制剂的制备方法。A method of preparing a Schizochyphyte preparation according to any one of claims 5-10.
  16. 下述任一产品：Any of the following products:
    Y1)用于培养权利要求1或2中所述的裂壶藻(Schizochytrium limacinum)的培养基，为权利要求6中所述发酵培养基；Y1) a medium for cultivating the Schizochytrium limacinum according to claim 1 or 2, which is the fermentation medium described in claim 6;
    Y2)权利要求1-10中任一所述制剂。Y2) The formulation of any of claims 1-10.
  17. 提高动物产品品质的方法，包括：饲喂动物裂壶藻(Schizochytrium limacinum)或其制剂实现提高所述动物产品品质。A method of improving the quality of an animal product, comprising: feeding an animal Schizochytrium limacinum or a preparation thereof to improve the quality of the animal product.
  18. 根据权利要求17所述的方法，其特征在于：所述提高动物产品品质为c1)和/或c2)和/或c3)：The method according to claim 17, characterized in that said improving the quality of the animal product is c1) and/or c2) and/or c3):
    c1)提高所述动物产品DHA含量；C1) increasing the DHA content of the animal product;
    c2)提高所述动物产品Sn-2位DHA含量；C2) increasing the DHA content of the animal product at the Sn-2 position;
    c3)降低所述动物产品胆固醇含量。C3) reducing the cholesterol content of the animal product.
  19. 根据权利要求17或18所述的方法，其特征在于：所述裂壶藻(Schizochytrium limacinum)为裂壶藻(Schizochytrium limacinum)HS01，所述裂壶藻(Schizochytrium limacinum)HS01在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.13746。The method according to claim 17 or 18, wherein the Schizochytrium limacinum is Schizochytrium limacinum HS01, and the Schizochytrium limacinum HS01 is preserved in Chinese microbial species. The deposit number of the General Microbiology Center of the Management Committee is CGMCC No. 13746.
  20. 根据权利要求17-19中任一所述的方法，其特征在于：所述动物为反刍动物，所述动物的所述裂壶藻(Schizochytrium limacinum)或其制剂饲喂量为d1)-d7)中的任一种：The method according to any one of claims 17 to 19, wherein the animal is a ruminant, and the animal is fed with a dose of d1)-d7) of Schizochytrium limacinum or a preparation thereof. Any of them:
    d1)50-500mg/天/头；D1) 50-500 mg / day / head;
    d2)50-250mg/天/头；D2) 50-250 mg / day / head;
    d3)75-250mg/天/头；D3) 75-250 mg / day / head;
    d4)100-250mg/天/头；D4) 100-250mg / day / head;
    d5)125-250mg/天/头；D5) 125-250 mg / day / head;
    d6)150-250mg/天/头；D6) 150-250mg / day / head;
    d7)200-250mg/天/头；D7) 200-250mg / day / head;
    或，所述动物为家禽，饲喂所述动物的食物中所述裂壶藻(Schizochytrium limacinum)或其制剂的质量含量为e1)-e3)中的任一种：Alternatively, the animal is a poultry, and the food of the animal is fed with any one of the mass content of the Schizochytrium limacinum or its preparation of e1)-e3):
    e1)0.5％-2.5％；E1) 0.5% - 2.5%;
    e2)0.5％-1.5％；E2) 0.5%-1.5%;
    e3)1％-1.5％。E3) 1% - 1.5%.
  21. 利用权利要求17-20中任一所述方法制备得到的动物产品或对所述动物产品加工得到的产品。An animal product prepared by the method of any one of claims 17-20 or a product obtained by processing the animal product.
  22. 根据权利要求21所述的产品，其特征在于：所述动物为b1)或b2)：The product of claim 21 wherein said animal is b1) or b2):
    b1)反刍动物；B1) ruminants;
    b1)牛；B1) cattle;
    所述动物产品为奶。The animal product is milk.
  23. 根据权利要求22所述的产品，其特征在于：所述对所述动物产品加工得到的产品为f1)-f6)中的任一种：The product according to claim 22, wherein said product processed from said animal product is any one of f1)-f6):
    f1)原生易吸收的DHA乳制品；F1) natively absorbable DHA dairy products;
    f2)原生DHA纯牛奶F2) Native DHA pure milk
    f3)原生DHA巴氏奶F3) Native DHA pasteurized milk
    f4)原生DHA酸奶F4) Native DHA yogurt
    f5)原生DHA奶粉F5) Native DHA milk powder
    f6)酸奶。F6) Yogurt.
  24. 根据权利要求21所述的产品，其特征在于：所述动物为a1)或a2)或a3)：The product according to claim 21, wherein said animal is a1) or a2) or a3):
    a1)家禽；A1) poultry;
    a2)鸡；A2) chicken;
    a3)北京白鸡、海兰白鸡、海兰褐蛋鸡或海兰粉壳鸡；A3) Beijing white chicken, Hailan white chicken, Hailan brown laying hen or Hailan powder shell chicken;
    所述动物产品为蛋。The animal product is an egg.
  25. 根据权利要求24所述的产品，其特征在于：所述对所述动物产品加工得到的产品为原生磷脂型DHA蛋制品。The product according to claim 24, wherein said product processed from said animal product is a native phospholipid-type DHA egg product.
  26. 提高动物产品产量的方法，包括：饲喂动物裂壶藻(Schizochytrium limacinum)或其制剂实现提高所述动物产品产量。A method of increasing the yield of an animal product comprising: feeding an animal Schizochytrium limacinum or a formulation thereof to increase the yield of the animal product.
  27. 根据权利要求26所述的方法，其特征在于：所述裂壶藻(Schizochytrium limacinum)为裂壶藻(Schizochytrium limacinum)HS01，所述裂壶藻(Schizochytrium limacinum)HS01在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.13746。The method according to claim 26, wherein the Schizochytrium limacinum is Schizochytrium limacinum HS01, and the Schizochytrium limacinum HS01 is in the Chinese Microbial Culture Collection Management Committee. The deposit number of the General Microbiology Center is CGMCC No. 13746.
  28. 根据权利要求26或27所述的方法，其特征在于：所述动物为家禽，饲喂所述动物的食物中所述裂壶藻(Schizochytrium limacinum)或其制剂的质量含量为e1)-e3)中的任一种：e1)0.5％-5％；e2)0.5％-1.5％；e3)1％-1.5％。The method according to claim 26 or 27, wherein the animal is poultry, and the mass of the Schizochytrium limacinum or the preparation thereof is e1)-e3) in the food for feeding the animal. Any of: e1) 0.5% - 5%; e2) 0.5% - 1.5%; e3) 1% - 1.5%.
PCT/CN2018/115327 2018-04-04 2018-11-14 Application of schizochytrium limacinum and preparation thereof in improvement of quality and yield of animal product WO2019192182A1 (en)

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